Amphiphilic compounds

ABSTRACT

Bringing membrane proteins into aqueous solution generally requires the use of detergents or other amphiphilic agents. The invention provides a new class of amphiphiles, each of which includes a multi-fused ring system as a lipophilic group. These new amphiphiles confer enhanced stability to a range of membrane proteins in solution relative to conventional detergents, leading to improved structural and functional stability of membrane proteins, including integral membrane proteins. Accordingly, the invention provides new amphiphiles for biochemical manipulations and characterization of membrane proteins. These amphiphiles display favorable behavior with membrane proteins and can be used to aid the solubilization, isolation, purification, stabilization, crystallization, and/or structural determination of membrane proteins.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to U.S.Provisional Patent Application No. 61/621,293, filed Apr. 6, 2012, whichis incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with government support under GM075913,GM083118, and NS028471 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

Integral membrane proteins (IMPs) are crucial cellular components,mediating the transfer of material and signals between the extracellularenvironment and the cytoplasm, or between different cellularcompartments. Structural and functional analysis of IMPs is important tofurthering our understanding of membrane protein interactions. More thanhalf of current pharmaceutical agents target proteins in this class. IMPcharacterization is often challenging, and sometimes impossible, becauseof difficulties associated with handling these macromolecules. IMPs inthe native state display large hydrophobic surfaces, which are notcompatible with an aqueous environment. Detergents are thereforerequired to extract IMPs from the lipid bilayer and to maintain thenative state of the protein in solution. Nonionic detergents, such asdodecyl-β-D-maltoside (DDM) and octyl-β-D-glucoside (OG), are commonlyused for these extractions. Despite the comparatively mild nature ofDDM, OG and related detergents, many membrane proteins denature and/oraggregate upon solubilization with these agents.

Diverse strategies have been pursued to develop new tools forsolubilization of IMPs from membranes and for maintenance of theseproteins in a native-like state in aqueous solution. Unfortunately,techniques that are effective for solubilization are not always optimalor effective for stabilization, and vice versa. Strategies fordeveloping new IMP tools have included exploration of small amphiphilicmolecules that depart from traditional detergent architectures. Smallamphiphiles that facilitate IMP crystallization are particularlynoteworthy (see Chae et al., Nat. Methods 2010, 7, 1003-1008; Hovers etal., Mol. Membr. Biol. 2011, 28, 170; Rasmussen, et al., Nature 2011,469, 236-240; Rosenbaum et al., Nature 2011, 469, 175-180; Rasmussen etal., Nature 2011, doi: 10.1038/nature10361).

Amphiphilic polymers (“amphipols”) and discoidal lipid bilayersstabilized by an amphiphilic protein scaffold (“nanodiscs”) representhighly innovative approaches for stabilizing IMPs in native-like statesin aqueous solution. It is not clear, however, whether either of theseapproaches can support growth of high-quality crystals for diffractionanalysis. Furthermore, neither amphipols nor nanodiscs were designed toextract IMPs from biological membranes. Despite considerable progress inthe development of new compounds and strategies for membrane proteinsolubilization and stabilization, new tools are needed, because manyIMPs are currently refractory. Given the great variation in structureand physical properties among membrane proteins, it is very unlikelythat a single amphiphile or amphiphile family will be optimal for everysystem, or even most systems.

Accordingly, there is a need for new classes of structurally novelamphiphiles that display favorable behavior, relative to traditionaldetergents such as DDM, toward a diverse set of membrane proteins. Thereis also a need for novel amphiphiles that can aid membrane proteinmanipulation techniques such as solubilization, isolation,stabilization, and crystallization.

SUMMARY

The invention provides new amphiphiles for protein manipulation. Themanipulation can include solubilization, stabilization, isolation,purification, crystallization and/or assistance in structuraldetermination of membrane proteins, such as difficult to solubilizeintegral membrane proteins. The new class of amphiphiles can bear rigidhydrophobic groups derived from steroid-like structures for manipulationof membrane proteins.

The invention thus provides a compound of Formula I:

wherein

L is —(CH₂)_(n)— where n is 1-12, or a direct bond;

X is NH, O, or a direct bond;

Y is O or absent;

Z is H, methyl, ethyl, propyl, or butyl;

R^(x) is H, optionally substituted (C₁-C₂₄)alkyl, optionally substitutedaryl or aroyl, or an oxygen-linked monosaccharide, disaccharide, ortrisaccharide; and

-   -   each Sac is independently an oxygen-linked monosaccharide,        disaccharide, or trisaccharide.

The variable R^(x) can be methyl, ethyl, propyl, or any optionallysubstituted (C₁-C₂₄)alkyl group. Each Sac can be an oxygen-linkedmonosaccharide, an oxygen-linked disaccharide, or an oxygen-linkedtrisaccharide. Various specific saccharides that can be attached toFormula I include those recited herein in the definition of saccharide.In some embodiments, each Sac group is a disaccharide moiety, such as amaltosyl group.

In some embodiments, X is NH, Y is O, Z is H, and L is a direct bond.

In other embodiments, L is —CH₂—, X is O, Y is absent, and Z is Me.

In yet other embodiments, L is a direct bond, X is a direct bond, Y isabsent, and Z is H.

The compound of Formula I can be a compound of Formula II, III, or IV:

wherein DiSac is an oxygen-linked disaccharide. In certain specificembodiments, the compound is:

In another embodiment, the invention provides a compound of Formula V:

wherein

L is —(CH₂)_(n)— where n is 1-12, or a direct bond;

X is NH or O;

Y is O or absent;

Z is H, methyl, ethyl, propyl, or butyl;

the dashed line represents an optionally present double bond; and

each Sac is independently an oxygen-linked monosaccharide, disaccharide,or trisaccharide.

The optional double bond can be present, or it can be absent, forexample if removed by hydrogenation or another reduction reaction.

Each Sac can be an oxygen-linked monosaccharide, an oxygen-linkeddisaccharide, or an oxygen-linked trisaccharide. Various specificsaccharides that can be attached to Formula I include those recitedherein in the definition of saccharide. In some embodiments, each Sacgroup is a disaccharide moiety, such as a maltosyl group.

In some embodiments, X is NH, Y is O, Z is H, and L is a direct bond.

In other embodiment, L is —CH₂—, X is O, Y is absent, and Z is Me.

In yet other embodiments, L is a direct bond, X is a direct bond, Y isabsent, and Z is H.

In further embodiments, the variable n of group L can be 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, or 12, for example, for either Formula I or FormulaV.

The compound of Formula V can be a compound of Formula VI:

wherein DiSac is an oxygen-linked disaccharide.

The compound of Formula VI can be, for example:

The stereochemistry of the above formulas and structures are merelyillustrative of certain embodiments. The invention can include otherstereoisomers including the enantiomers and various diastereomers of theformulas and structures shown.

In some embodiments, the critical micelle concentration (CMC) of acompound of Formula I-VI in water is about 5 nM to about 100 nM. Invarious embodiments, the CMC can be about 5 nM to about 60 nM, about 5nM to about 20 nM, or about 45 nM to about 55 nM. A plurality of thecompounds can form a micelle in water comprising about 5 to about 35molecules of the compound. Some micelles can include about 5 to about10, about 10 to about 15, about 5 to about 20, about 5 to about 15,about 10 to about 20, or about 5 to about 25 molecules of the compoundin the formation of individual micelles.

The invention also provides a composition comprising a plurality ofcompounds as described above and an isolated membrane protein. Thecomposition can include micelles that include the compounds describedherein encapsulating the isolated membrane protein, optionally incombination with other compounds, amphiphiles, or surfactants in themicelle structure. The micelle can optionally include one or more drugs,therapeutic molecules, bioactive molecules, polypeptides, proteins,genes, or a combination thereof, within the micelle. In someembodiments, the molecule within the micelle is a polypeptide or aprotein.

The invention also provides methods of solubilizing or stabilizing amembrane protein comprising contacting a membrane protein with aneffective amount of a plurality of compounds described herein, in anaqueous solution. The methods can and optionally include heating theprotein and the compounds, thereby forming a solubilized or stabilizedaggregation or micelle of the compounds and the membrane protein. Theinvention further provides methods of extracting a protein from a lipidbilayer comprising contacting the lipid bilayer with an effective amounta plurality of compounds described herein in an aqueous solution orsuspension to form a mixture, optionally in the presence of a buffer,thereby forming an aggregation or micelle of the compounds and themembrane protein that has been extracted from the lipid bilayer. Theaggregates and/or micelles can then be separated from the mixture toprovide the isolated proteins. The compounds described herein can beparticularly valuable for stabilizing proteins in a functional form suchthat the protein can be analyzed by various assays, such as a ligandbinding assay.

The invention therefore provides novel compounds and formulas,intermediates for the synthesis of the compounds and formulas, as wellas methods of preparing the compounds, formulas, and compositionsdescribed herein. The invention also provides compounds that are usefulas intermediates for the synthesis of other valuable compounds. Theinvention further provides methods of using the compounds, for example,to aid the solubilization, isolation, purification, stabilization,crystallization, and/or structural determination of membrane proteins.The compounds of the invention can be used alone, or in combination withlipids or known detergents. Other objects, features and advantages ofthe present invention will become apparent from the followingdescription, claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the specification and are includedto further demonstrate certain embodiments or various aspects of theinvention. In some instances, embodiments of the invention can be bestunderstood by referring to the accompanying drawings in combination withthe detailed description presented herein. The description andaccompanying drawings may highlight a certain specific example, or acertain aspect of the invention. However, one skilled in the art willunderstand that portions of the example or aspect may be used incombination with other examples or aspects of the invention.

FIG. 1. Chemical structures of new amphiphiles GLC-1, GLC-2, GLC-3, andGDN.

FIG. 2. Stability of (a) bR and (b) R. capsulatus LHI-RC superassemblyat RT as a function of time. Agents were tested at 0.2 wt % OTG+1.6 wt %amphiphile for bR and at CMC+0.04 wt % for the R. capsulatussuperassembly.

FIG. 3. Time course of bacteriorhodopsin (bR) stability at RT. OTG wasmixed with each agent in a ratio of (a) 1:4 (0.2 wt % OTG+0.8 wt %GLCs/GDN) or (b) 1:8 (0.2 wt % OTG+1.6 wt % GLCs/GDN). Absorbance at 554nm was followed for the long-term stability evaluation of the protein.

FIG. 4. Time course of LHI-RC complex stability at RT. The R. capsulatussuperassembly was purified with each GLC or GDN at three differentconcentrations: (a) CMC+0.04 wt %, (b) CMC+0.20 wt % and (c) CMC+1.0 wt%. Absorbance ratio (A₈₇₅/A₆₈₀) was used as an indicator ofsuperassembly integrity.

FIG. 5. (a, b) Gel filtration analysis for CMP-Sia and (c) activity overtime of LeuT (scintillation proximity assay (SPA), based on [³H]-Leubinding). Gel filtration analysis was performed at a detergentconcentration of CMC+0.04 wt %, before or after incubation ofsolubilized CMP-Sia at 30° C. for 2 hr. SPA was conducted withdetergents at CMC+0.04 wt % or CMC+0.2 wt % with LeuT stored at RT. SPAresults are expressed as % activity relative to the day 0 measurements.

FIG. 6. CPM assays for (a) CMP-Sia, (b) GlpG and (c) SQR solubilizedwith each new amphiphile or DDM. The coumarin moiety of CPM isinternally quenched by the maleimide unit, but the coumarin becomesfluorescent following reaction with Cys side chain thiol groups exposedupon protein unfolding. The CPM assay can therefore be used to monitorthe extent of protein unfolding. CMP-Sia and GlpG were initiallyextracted from the native membrane with 1% DDM in PBS, 10 mM imidazole(pH 8.0), 150 mM NaCl, 10% glycerol, and isolated in 20 mM Tris (pH7.5), 150 mM NaCl containing 0.03% DDM. SQR was extracted from themembrane using 2% C₁₂E₉ in 20 mM potassium phosphate (pH 7.4), 0.2 MEDTA, and isolated in 20 mM Tris (pH 7.6), 0.2% decyl-β-D-maltoside(DM). The purified proteins (CMP-Sia (6 mg/ml), GlpG (5 mg/ml) and SQR(12 mg/ml)) were diluted 1:150 in 20 mM Tris (pH 7.5), 150 mM NaClcontaining CMC+0.04 wt % amphiphile or DDM. The CPM analysis wasperformed over 130 min at 30° C. using a microplate spectrofluorometerset at an excitation wavelength of 387 nm and an emission wavelength of463 nm. Measurements were taken every 5 min after automatic agitation ofthe plate. The vertical axes in these graphs have no absolute meaning.The “Relative amount of folded protein” in each case is defined asfollows: 100% corresponds to the fluorescence emission intensity attime=0 min; 0% corresponds to the lowest value measured among theamphiphile-treated samples for each protein during the 130 min assayperiod. Thus, for CMP-Sia, 0% is defined by the end-point measurementfor protein solubilized with GLC-1. For GlpG and SQR, 0% is defined bythe lowest value measured for protein solubilized with DDM. In no casecan the “0%” value be interpreted as indicating that the protein isfully unfolded. This point is demonstrated by the gel filtration resultsshown for CMP-Sia in the description below (FIG. 5), which indicate that˜50% of the protein solubilized with DDM remains intact at the end ofthe incubation period; however, in FIG. 6 a, DDM-solubilized CMP-Sia isindicated to contain ˜20% “relative amount of folded protein” under theconditions used for the gel filtration analysis.

FIG. 7. Time course activity (scintillation proximity assay, SPA, basedon [³H]-Leu binding) for LeuT solubilized with GLC amphiphiles (GLC-1,GLC-2 or GLC-3) or DDM at (a) CMC+0.04 wt % and (b) CMC+0.20 wt %. SPAwas conducted on protein stored at RT. Results are expressed as % ofactivity relative to the day 0 measurements (mean±s.e.m., n=2).

FIG. 8. a) Melting temperatures (T_(m)) of β₂AR-T4L, and b) β₂AR WTactivity as a function of time, for proteins solubilized with newamphiphiles or DDM, demonstrating long-term stability properties of β₂ARstabilized by the new amphiphiles. T_(m) values for β₂AR-T4L are plottedin terms of wt % of the amphiphile. β₂AR WT was extracted with 1 wt % or2 wt % amphiphile, and activity was measured periodically byradioligand-binding assay using the antagonist [³H]-dihydroalprenolol.The solubilized β₂AR WT samples were stored at 4° C.

FIG. 9. Activity of δ-opioid receptor-T4L (δOR-T4L) solubilized withDDM, MNG-3 or GDN. The receptor was extracted with 1.0 wt % ofamphiphile, and ligand binding activity (counts per minute (cpm)) wasmeasured by radioligand-binding assay using the antagonist[³H]-diprenorphine.

FIG. 10. SDS-12% PAGE and Western blot analysis of MelB. Samples wereanalyzed by SDS-PAGE analysis, and MelB was detected usinganti-histidine tag antibody. Each sample contained 10 μg protein. Forextracts generated with each detergent or amphiphile at eachtemperature, one sample was subjected to ultracentrifugation (+), and acomparison sample was not (−). As a control, an untreated membranesample (“Memb.”; no ultracentrifugation) was included in each gel.

FIG. 11. SDS-12% PAGE and Western blot analysis of MelB-EC. MelB-ECprotein was expressed in E. coli and treated with DDM or GDN forextraction. The samples were then separated by SDS-PAGE analysis, anddetected by western blotting using anti-histidine tag antibody. Eachsample included 10 μg proteins. For extracts with each detergent oramphiphile, one sample was subjected to ultracentrifugation (+) and acomparison sample was not (−). As a control, an untreated membranesample (“Memb.”; no ultracentrifugation) was included in each gel.

FIG. 12. The characterization of (a) LHI-RC complex and (b) β₂AR WTextracted with GDN or conventional detergents (DDM,laurydimethylamine-N-oxide (LDAO), and n-octyl-β-D-glucopyranoside(OG)). The superassembly amount was estimated via spectrophotometry andβ₂AR WT was detected by western blotting using M1 antibody.

FIG. 13. Synthetic scheme for the preparation of GLC-1 and GLC-2.Reagents and conditions: (a) EDC.HCl, HOBt, DMF, RT, 2 days; (b)perbenzoylated maltosylbromide, AgOTf, CH₂Cl₂, −45° C.→RT, 3 hr; (c)NaOMe, MeOH, RT, 4 hr; (d) LiAlH₄, THF, RT, 1 day; (e) CBr₄, Ph₃P,MeCN:THF, RT, 15 hr; (f) 1,1,1-Tris(hydroxymethyl)ethane, NaH, 60° C., 2hr.

FIG. 14. Synthetic scheme for the preparation of GLC-3. Reagents andconditions: (b) perbenzoylated maltosylbromide, AgOTf, CH₂Cl₂, −45°C.→RT, 3 hr; (c) NaOMe, MeOH, RT, 4 hr; (d) LiAlH₄, THF, RT, 1 day; (e)CBr₄, Ph₃P, MeCN:THF, RT, 15 hr; (g) diethylmalonate, NaH, THF, RT, 15hr; LiAlH₄, THF, RT, 1 day.

FIG. 15. Synthetic scheme for the preparation of the amphiphile GDN.Reagents and conditions: (a) LiAlH₄, THF, RT, 1 day; (b) CBr₄, Ph₃P,CH₂Cl₂, RT, 15 hr; (c) diethylmalonate, NaH, THF, RT, 1 day; LiAlH₄,THF, RT, 1 day; (d) perbenzoylated maltosylbromide, AgOTf, CH₂Cl₂, −45°C.→RT, 3 hr; (e) NaOMe, MeOH, RT, 4 hr.

DETAILED DESCRIPTION

The difficulty of obtaining crystal structures for membrane proteinsrepresents a profound hindrance to fundamental and applied biologicalresearch. Many MPs cannot be maintained in native-like conformationswhen solubilized with conventional detergents. Moreover, even when anative conformation can be achieved, the MP-detergent complex withtraditional detergents such as DDM can manifest unfavorable propertieswith regard to structural analysis. The complexes may have the inabilityto crystallize, and/or the complexes formed may be too large foreffective NMR analysis). Because our understanding of membrane proteinstructure and function remains poorly developed relative to solubleproteins, there is a persistent need for new amphiphilic “assistants”that can promote solubilization and manipulation of MPs.

Bringing membrane proteins into aqueous solution generally requires theuse of a detergent or other amphiphilic agents. Disclosed herein is anew class of amphiphiles, each of which uses a multi-fused ring systemas a lipophilic group. This family of molecules confers enhancedstability to a range of membrane proteins in solution relative toconventional detergents, leading to improved structural and functionalstability of integral membrane proteins (IMPs).

Analyses of the new amphiphiles indicate they are comparable or superiorto the commonly used biochemical detergent DDM with respect to severaldifferent protein systems. These results indicate that the newamphiphiles are at least complementary to current technology, such asknown commercial biochemical detergents, in the context of many membraneproteins that researchers would like to study. In general, a significantfraction of these proteins of interest remain very difficult to examine,and so new amphiphiles with distinctive structures and properties, suchas those described herein, will be attractive as research tools.

A large number of amphiphiles are needed on the market forcharacterization and solubilization work, because many alternatives mustbe tried for each membrane protein to identify the best match.Accordingly, the amphiphiles described herein will provide additionalresources to researchers for manipulating membrane proteins. Forexample, the amphiphiles can be used as reagents for proteinsolubilization and crystallization, especially for generally insolubleproteins. The amphiphiles can also be used as reagents for proteinstabilization, so that the proteins can be analyzed by various ligandbinding assays. For a continuously updated database of MP structures,each of which can be potentially suitably manipulated by the amphiphilesdescribed herein, see:http://blanco.biomol.uci.edu/Membrane_Proteins_xtal.html.

Before the amphiphiles and methods are further described, it is notedthat this invention is not limited to the particular methodology,protocols, materials, and reagents described, as these may vary. Forexample, various sugars, disaccharides and trisaccharides can beexchanged for other isomers in the preparation of the compounds. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only and is not intended tolimit the scope of the invention, which is limited only by the appendedclaims.

Definitions

As used herein, the recited terms have the following meanings. All otherterms and phrases used in this specification have their ordinarymeanings as one of skill in the art would understand. Such ordinarymeanings may be obtained by reference to technical dictionaries, such asHawley's Condensed Chemical Dictionary 14^(th) Edition, by R. J. Lewis,John Wiley & Sons, New York, N.Y., 2001.

References in the specification to “one embodiment”, “an embodiment”,etc., indicate that the embodiment described may include a particularaspect, feature, structure, moiety, or characteristic, but not everyembodiment necessarily includes that aspect, feature, structure, moiety,or characteristic. Moreover, such phrases may, but do not necessarily,refer to the same embodiment referred to in other portions of thespecification. Further, when a particular aspect, feature, structure,moiety, or characteristic is described in connection with an embodiment,it is within the knowledge of one skilled in the art to affect orconnect such aspect, feature, structure, moiety, or characteristic withother embodiments, whether or not explicitly described.

The singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, a referenceto “a compound” includes a plurality of such compounds, so that acompound X includes a plurality of compounds X. It is further noted thatthe claims may be drafted to exclude any optional element. As such, thisstatement is intended to serve as antecedent basis for the use ofexclusive terminology, such as “solely,” “only,” and the like, inconnection with the recitation of claim elements or use of a “negative”limitation.

The term “and/or” means any one of the items, any combination of theitems, or all of the items with which this term is associated. Thephrase “one or more” is readily understood by one of skill in the art,particularly when read in context of its usage. For example, one or moresubstituents on a phenyl ring refers to one to five, or one to four, forexample if the phenyl ring is disubstituted.

The term “about” can refer to a variation of ±5%, ±10%, ±20%, or ±25% ofthe value specified. For example, “about 50” percent can in someembodiments carry a variation from 45 to 55 percent. For integer ranges,the term “about” can include one or two integers greater than and/orless than a recited integer at each end of the range. Unless indicatedotherwise herein, the term “about” is intended to include values, e.g.,weight percents, proximate to the recited range that are equivalent interms of the functionality of the individual ingredient, thecomposition, or the embodiment.

As will be understood by the skilled artisan, all numbers, includingthose expressing quantities of ingredients, properties such as molecularweight, reaction conditions, and so forth, are approximations and areunderstood as being optionally modified in all instances by the term“about.” These values can vary depending upon the desired propertiessought to be obtained by those skilled in the art utilizing theteachings of the descriptions herein. It is also understood that suchvalues inherently contain variability necessarily resulting from thestandard deviations found in their respective testing measurements.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges recited herein also encompass any and all possible sub-ranges andcombinations of sub-ranges thereof, as well as the individual valuesmaking up the range, particularly integer values. A recited range (e.g.,weight percents or carbon groups) includes each specific value, integer,decimal, or identity within the range. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths, ortenths. As a non-limiting example, each range discussed herein can bereadily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art, all languagesuch as “up to”, “at least”, “greater than”, “less than”, “more than”,“or more”, and the like, include the number recited and such terms referto ranges that can be subsequently broken down into sub-ranges asdiscussed above. In the same manner, all ratios recited herein alsoinclude all sub-ratios falling within the broader ratio. Accordingly,specific values recited for radicals, substituents, and ranges, are forillustration only; they do not exclude other defined values or othervalues within defined ranges for radicals and substituents.

One skilled in the art will also readily recognize that where membersare grouped together in a common manner, such as in a Markush group, theinvention encompasses not only the entire group listed as a whole, buteach member of the group individually and all possible subgroups of themain group. Additionally, for all purposes, the invention encompassesnot only the main group, but also the main group absent one or more ofthe group members. The invention therefore envisages the explicitexclusion of any one or more of members of a recited group. Accordingly,provisos may apply to any of the disclosed categories or embodimentswhereby any one or more of the recited elements, species, orembodiments, may be excluded from such categories or embodiments, forexample, as used in an explicit negative limitation.

The term “contacting” refers to the act of touching, making contact, orof bringing to immediate or close proximity, including at the cellularor molecular level, for example, to bring about a physiologicalreaction, a chemical reaction, or a physical change, e.g., in asolution, in a reaction mixture, in vitro, or in vivo.

The phrase “treating a protein” with a compound, detergent, surfactant,or “agent” refers to contacting the protein with the agent (e.g., anamphiphile as described herein), and/or combining the protein with aneffective amount of the agent under conditions that allow the agent topenetrate, integrate and/or disrupt a protein's current environment inorder to solubilize, stabilize, isolate, and/or purify the protein. Theconditions can be aqueous and additional reagents, such as buffers,salts, and the like, can be added. The treating can use a single type ofagent, such as an amphiphile described herein, or the treating canemploy a combination of agents, such as an amphiphile described hereinin combination with one or more surfactants such as DDM, CHAPS, CHAPSO,and the like. Thus, a combination of reagents may be employed in thetreatment. The protein may be, for example, in a lipid bilayer orsubstantially isolated in solution.

Detergent-solubilized membrane proteins are typically more thermolabilethan their membrane-embedded forms, therefore stabilizing a protein isimportant for research and analysis. The phrase “stabilizing a protein”refers to treating a protein so that the protein thermostabilityimproves, or so that the protein retains activity (e.g., of a particularreceptor), or maintains a native confirmation, for example, whenextracted from a membrane. Stabilizing a membrane protein with anamphiphile as described herein can be, for example, improving its T₅₀value by about 5° C., about 10° C., about 15° C., about 20° C., or about25° C., for example, compared to a standard detergent such as DDM.Increasing the stability of an isolated protein is important to allowresearchers sufficient time to examine and characterize the protein.

Methods of the invention include treating a protein, for example, usingsuch techniques as solubilization, isolation, purification,stabilization, crystallization, and/or structural determination. Themethods can include standard laboratory techniques such as lysing acell, precipitation, concentration, filtration, and/or fractionation.

An “effective amount” refers to an amount effective to bring about arecited effect. For example, an amount effective can be an amount of anagent or combination of reagents effective to solubilize or stabilize amembrane protein.

The term “alkyl” refers to a branched or unbranched hydrocarbon having,for example, from 1-20 carbon atoms, and often 1-12, 1-10, 1-8, 1-6, or1-4 carbon atoms. Examples include, but are not limited to, methyl,ethyl, 1-propyl, 2-propyl(iso-propyl), 1-butyl,2-methyl-1-propyl(isobutyl), 2-butyl(sec-butyl),2-methyl-2-propyl(t-butyl), 1-pentyl, 2-pentyl, 3-pentyl,2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl,1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl,4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl,2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, hexyl, octyl, decyl,dodecyl, and the like. The alkyl can be a monovalent hydrocarbonradical, as described and exemplified above, or it can be a divalenthydrocarbon radical (i.e., an alkylene), according to the context of itsusage.

The term “aroyl” refers to the group aryl-C(═O)—. Examples of arylgroups include benzyl, anthryl, biphenyl, and the like. The aryl groupsof the aroyl can be optionally substituted with substituents such asalkyl, halo (F, Cl, Br, or I), nitro, amino, and the like.

The term “saccharide” refers to a sugar or sugar moiety, such as amonosaccharide, a disaccharide, or a trisaccharide. Typicalmonosaccharides include allose, altrose, glucose, mannose, gulose,idose, galactose, or talose. Typical disaccharides include galactose,lactose, maltose, sucrose, trehalose, and cellobiose. Disaccharides canhave any suitable linkage between the first and the second unit of thedisaccharide. Other suitable saccharides include glucuronic acid,sorbase, ribose, and the like. A saccharide can include hydroxylprotecting groups such as, but not limited to, acetyl groups, benzylgroups, benzylidene groups, silyl groups, methoxy ether groups, orcombinations thereof. The saccharide groups can also be in pyranoseform, furanose form, or linear form. The saccharides can be linked toFormula described herein via their anomeric oxygen, or to any otheravailable hydroxyl group. Depending on the context, as would beunderstood by one of skill in the art, the saccharide can include theoxygen that links it to another group, or exclude the oxygen that linksit to another group.

Trisaccharides are oligosaccharides composed of three monosaccharideswith two glycosidic bonds connecting them. Similar to disaccharides,each glycosidic bond can be formed between any hydroxyl group on thecomponent monosaccharides. The three monosaccharide components can havedifferent bond combinations (regiochemistry) and stereochemistry (alpha-or beta-linkages) to provide trisaccharides that are variousdiastereomers. Examples of trisaccharides include nigerotriose,maltotriose, maltotriulose, and raffinose.

The saccharides described herein may include one or morehydroxylprotecting groups, such as benzyl groups, acetyl groups, orbenzoyl groups. However, some embodiments have most or all of theprotecting groups removed from the saccharide groups of the amphiphilesdescribed herein.

The “Critical Micelle Concentration” (CMC) refers to the concentrationof a detergent (e.g., an amphiphile as described herein) in an aqueoussolution at which the detergent molecules self-assemble into micelles.Below the CMC, detergents are mostly monomeric; above the CMC, micelleconcentration increases linearly with detergent concentration. The CMCis dependent upon many factors and is detergent-specific. The CMC of adetergent can be determined experimentally by measuring thesolubilization of a water-insoluble dye or fluorophore while varying theconcentration of detergent. A CMC may also be determined by measuringthe diminution of the surface tension of an aqueous solution as afunction of detergent concentration (CMCs determined by either methodcorrelate with each other). The CMC is determined by extrapolating theplot of solubilization vs. concentration (or surface tension vs.concentration) in the two linear regions above and below the CMC. Wherethe two lines intersect is the CMC. The CMC can also be determined bythe method of Nugebauer, J. M. (1990), Methods in Enzymology,182:239-253.

Amphiphiles for Membrane Protein Manipulation

The new amphiphiles can contain a rigid, steroid-based lipophilic groupand a pair of saccharides to provide a hydrophilic group. Four specificexamples are illustrated in FIG. 1. Three of the new compounds arederived from lithocholic acid and are therefore designated“glyco-lithocholate” amphiphiles (GLC-1, GLC-2 and GLC-3); the fourth isderived from diosgenin and is designated “glyco-diosgenin” (GDN).Previously reported amphiphiles based on steroidal skeletons have beenderivatives of cholic acid or deoxycholic acid, including members of thewidely-used CHAPS family, cholate-based facial amphiphiles, andtandem-facial amphiphiles (Hjelmeland, Proc. Natl. Acad. Sci. USA 1980,77, 6368-6370; Zhang et al., Angew. Chem. Int. Ed. 2007, 46, 7023-7025;Chae et al., J. Am. Chem. Soc. 2010, 132, 16750-16752). In these casesthe rigid steroidal units are facially amphiphilic: one side ishydrophilic, displaying either hydroxyl groups or carbohydrate units. Incontrast, the hydrophobic units in the GLC and GDN amphiphilesintroduced here are hydrophobic on both faces, and the hydrophilicmoiety is appended to the periphery of the rigid hydrophobic unit.

The four compounds were readily prepared on a multi-gram scale (seeExamples 1 and 2 below, and FIGS. 13-15), as is necessary if they are toserve as research tools. All four are highly soluble in aqueous solution(>20 wt %) and have relatively low critical micelle concentrations (CMC;determined by fluorescent dye solubilization). See Table 1.

TABLE 1 Critical Micelle Concentration (CMC) of GLC/GDN amphiphiles andhydrodynamic radii (Rh) of their micelles (Mean ± SD, n = 5). MW^([a])CMC (μM) CMC (wt %) R_(h) (nm)^([b]) GLC-1 1112.3 ~52 ~0.006 3.22 ± 0.03GLC-2 1127.3 ~8.0 ~0.0009 3.32 ± 0.04 GLC-3 1083.3 ~7.1 ~ 0.00077 3.27 ±0.08 GDN 1165.3 ~18 ~0.0021 3.86 ± 0.05 DDM 510.1 ~170 ~0.0087 3.42 ±0.03 ^([a])Molecular weight of detergents. ^([b])Hydrodynamic radius ofmicelles measured by dynamic light scattering.

These values are somewhat smaller than the CMC of DDM, which indicates astrong tendency of the new agents to self-assemble, and which can beadvantageous for IMP handling because detrimental levels of non-micellaramphiphile can be avoided. Table 1 provides the hydrodynamic radius (Rh)of the micelles formed by each amphiphile, as determined by dynamiclight scattering (DLS). The micelles formed by GLC amphiphiles areslightly smaller than those formed by DDM, while the micelles formed byGDN are slightly larger.

Thus, the amphiphiles described herein can be prepared from commerciallyavailable steroidal precursors such as cholesterol and relatedmolecules. The steroid precursor compound can be rendered water-solubleby attachment of a bis-saccharide unit, such as a bis-maltoside unit.The amphiphiles generally fall into two classes, GLCs and GDNs (see FIG.1). These amphiphilic molecules can be used as tools in biochemistry,specifically for solubilization, stabilization and crystallization ofmembrane proteins.

For example, amphiphiles can be prepared as illustrated in FIGS. 13-15.The acid functionality of lithocholic acid can be converted to an amide,ether, or alkylene linking group, which can then be glycosylated withvarious saccharides, such as maltosyl groups, or other saccharidesrecited herein. In other embodiments, the free hydroxyl of diosgenin canbe functionalized similarly with ether, or alkylene linking group, whichcan also be glycosylated as described herein. The protected saccharidegroups can then be deprotected or partially deprotected to providevarious amphiphiles of the invention.

The saccharide moieties can be maltose, as illustrated in FIGS. 13-15,or they can be other saccharides, such as one or more of themonosaccharides, disaccharides, or trisaccharides recited herein. Thesaccharides can include various protecting groups, as would be wellunderstood by one of skill in the art. Specific protecting groupsinclude benzyl, acetyl, trifluoroacetyl, benzoyl, benzyloxycarbonyl, andsilicon protecting groups such as trimethylsilyl, t-butyldimethylsilyl,and diphenylmethylsilyl. Other suitable protecting groups are known tothose skilled in the art (see for example, T. W. Greene, ProtectingGroups In Organic Synthesis; Wiley: New York, Third Edition, 1999, andreferences cited therein.

The synthetic transformations described above are well known in the artand are generally described by reference works such as J. March,Advanced Organic Chemistry, Reactions, Mechanisms and Structure, (2ndEd.), McGraw Hill: New York, 1977; Greg T. Hermanson in BioconjugateTechniques (Academic Press, San Diego, Calif. (1996)); and F. Carey andR. Sundberg, Advanced Organic Chemistry, Part B: Reactions andSynthesis, 2^(nd) Ed., Plenum: New York, 1977; and references citedtherein. Other useful synthetic techniques are described in U.S. Pat.No. 6,172,262 (McQuade et al.) and U.S. Patent Publication Nos.2009/0270598 (Gellman et al.) and 2010/0311956 (Gellman et al.).

Amphiphile Applications to Proteins and Membranes

Manipulation of membrane proteins remains a profound technicalchallenge. A variety of different amphiphiles are needed on the market,as different amphiphiles are useful for different target proteins,depending on the properties of the protein and the in vitro useproposed. The best amphiphile for any particular protein is difficult orimpossible to predict, and requires empirical testing. Researchers mostoften cannot predict which amphiphile will be suitably effective formanipulating a particular membrane protein. Data acquired for the newamphiphiles shows that they are comparable or superior to knowndetergents for membrane protein manipulation. Therefore the newamphiphiles described herein provide additional valuable tools for themanipulation of membrane proteins.

The invention provides compounds and compositions that can include aplurality of amphiphilic compounds described herein and a membraneprotein, such as an integral membrane protein. Such compositions cantake the form of aggregates or micelles, formed from a pluralityamphiphilic compounds as described herein, optionally in conjunctionwith one or more other surfactant compounds and/or micelle-formingcompounds, where the plurality of compounds surround the membraneprotein. The composition can optionally include a polypeptide, aprotein, and/or one or more other types of biological moleculescomplexed with the amphiphilic compound.

The invention thus provides methods of solubilizing a membrane proteinby contacting the membrane protein with a plurality of a compounddescribed herein, in an aqueous solution, thereby forming a solubilizedaggregation of the compounds and the membrane protein. The inventionalso provides methods of stabilizing a membrane protein by contactingthe membrane protein with a plurality of a compound described herein, inan aqueous solution, thereby forming an aggregation of the compounds andthe membrane protein. The invention further provides methods ofextracting a protein from a lipid bilayer by contacting the lipidbilayer with a plurality of a compound described herein in an aqueoussolution to form a mixture, optionally in the presence of a buffer orother detergent, thereby forming an aggregation of the compounds and themembrane protein extracted from the lipid bilayer. The aggregation canthen be separated from the mixture to provide isolated and/or purifiedmembrane protein.

Accordingly, the invention provides various methods for manipulatingmembrane proteins. For example, a method is provided for solubilizing amembrane protein by contacting the protein in an aqueous environmentwith an effective amount of a compound as described herein, andoptionally heating the protein and the compound, to provide thesolubilized protein encapsulated in micelles of the compound. Theeffective amount of the compound can be an amount of the compoundnecessary to achieve its critical micelle concentration, to about 10times, about 100 times, about 1,000 times, or about 10,000 times, theamount of the compound necessary to achieve its critical micelleconcentration. The method can also include employing a buffer, heat, asecond amphiphile or detergent, or other reagents, in the aqueousenvironment to aid in the solubilization and stabilization of membraneproteins.

The invention also provides a method of purifying a membrane protein bycontacting the protein in an aqueous environment with an effectiveamount of a compound as described herein, to form micelles comprising aplurality of the compounds surrounding the protein, and isolating themicelles, to provide the purified membrane protein encapsulated inmicelles of the compound. Other techniques for using the amphiphiliccompounds described herein include techniques for stabilizing,crystallizing, and/or characterizing a protein while in a detergentmicelle made up of a compound described herein.

The invention has several advantages over previous membrane manipulationtechnologies. For example, the amphiphiles described herein can lack anyaromatic groups, therefore they are highly suitable for “optical”characterization methods such as UV absorbance spectroscopy and UVcircular dichroism, when characterizing a protein solubilized by suchamphiphiles.

Other uses of the amphiphiles described herein include their use asamphiphilic additions in crystallization trials, components of detergentmixtures, stabilizing factors in functional assays, detergents inexchange schemes, solubilization agents in cell-free expressionreactions, as well as their use for separation on polyacrylamide gelsusing native protocols to maintain native states, for use in samplebuffers on membrane fractions used to solubilize membrane proteins andto prepare proteins for separation on gels, and for use with Bug Buster®Protein Extraction Reagent formulations designed to break open cells andsurvey protein present, for example, without using sonication and/orlysozyme treatment and osmotic shock, such as with eukaryotic cellpellets that are relatively fragile and easily disrupted.

The amphiphiles described herein can also aid the formation ofwell-ordered crystals of membrane protein-amphiphile complexes. When amembrane protein-amphiphile complex crystallizes, amphiphiles can beincluded within the crystal lattice or in other embodiments, excludedfrom the crystal lattice. The amphiphiles can contribute to the orderingof proteins within the lattice when crystals are formed, thereby aidingthe stability of growing membrane protein crystals.

The amphiphiles can stabilize membrane proteins, such as integralmembrane proteins, in native conformations, for example, for proteinstructural characterization. The amphiphiles can extract proteins fromlipid bilayers and stabilize the protein comparably or more effectivelythan conventional biological detergents. The amphiphiles can further beused for membrane protein research including isolation, stabilization,analysis by solution NMR, and biochemical and biophysical assaydevelopment.

The invention can therefore be directed to amphiphiles that can enhancethe ability of a composition to solubilize and crystallizemembrane-bound proteins into well-order crystals. The amphiphilesdescribed herein can be used in any application where conventionaldetergents are used. For instance, the amphiphiles can be used to lysecellular membranes. The amphiphiles can also form micelles in an aqueoussolution. They can therefore be used to solubilize hydrophobic compoundsfor dispersion into aqueous solution. More specifically, the amphiphilesare useful for solubilizing membrane proteins, such as integral membraneproteins.

The amphiphiles described herein can be used alone, or in combinationwith other biological detergents, such as DDM, undecyl-β-D-maltoside(β-UDM), 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate(CHAPS),3-[(3-cholamidopropyl)-dimethylammonio]-2-hydroxy-1-propanesulfonate(CHAPSO), lauryldimethylamine oxide (LDAO), octyl-glucoside (OG) orother detergents described by Hjelmeland in Methods of Enzymology, Vol.124, page 135-164, which is incorporated herein by reference. Forexample, a particular detergent may to too harsh to suitable solubilizea membrane protein in its native conformation, however a combination ofan amphiphiles described herein and a commercial biological detergentscan provide reduced severity, thereby allowing the protein to bemaintained in its native conformation while maintaining solubility.

Membrane Protein Manipulation

To assess the potential utility of new amphiphiles as tools for IMPmanipulation, multiple protein systems were examined. DDM was used as abenchmark for conventional detergent performance in each case becauseDDM is very widely employed for IMP studies. Bacteriorhodopsin (bR) hasbeen commonly used for evaluation of novel amphiphiles because stabilitycan be assessed conveniently via spectrophotometry (Schafineister etal., Science 1993, 262, 734-738; McQuade et al., Angew. Chem. 2000, 112,774-777; Angew. Chem. Int. Ed. 2000, 39, 758-761).

bR was extracted from the native purple membrane with 2.0 wt %octyl-β-D-thioglucoside (OTG) (see Bazzacco at al., Biomacromolecules2009, 10, 3317-3326). Following ultracentrifugation to remove insolubledebris, the bR solution was diluted with amphiphile solutions to give0.2 wt % OTG+1.6 wt % new agent or DDM. The absorbance of the solutionsat 554 nm was measured periodically over 20 days. FIG. 2 a shows thattwo new agents, GLC-2 and GDN, are more effective than conventionaldetergents OTG and DDM at maintaining the native structure (see FIG. 3for results with other agents). GDN provided exceptional stabilizationproperties, showing negligible loss in protein integrity after 20 days.When the assay was conducted at a lower amphiphile concentration, 0.2 wt% OTG+0.8 wt % new agent or DDM, similar results were obtained (FIG. 3).

Next a more challenging system, the photosynthetic superassembly fromRhodobacter capsulatus (Laible et al., Biochemistry 2003, 42,1718-1730), was analyzed. This photosynthetic superassembly contains thelight harvesting I (LHI) complex and the reaction center (RC) complex.The superassembly contains >30 protein molecules; integrity can beassessed based on the 875 nm/680 nm absorbance ratio (Chae et al.,ChemBioChem 2008, 9, 1706-1709). The superassembly was extracted fromthe native membrane with 1.0 wt % DDM and purified with DDM at its CMC(0.009 wt %). This preparation was diluted with solutions containing anew agent described herein, so that residual DDM (0.0004 wt %) was farbelow its CMC. The final concentration of each agent was CMC+0.04 wt %.FIG. 2 b shows that the LHI-RC superassembly is substantially morestable over 20 days when solubilized by GLC-2 or GDN relative tosolubilization with DDM or OG. Similar results were obtained withdifferent detergent concentrations (FIG. 4).

The promising behavior manifested by GDN in terms of the stability of bRand the R. capsulatus superassembly prompted the examining thisamphiphile with another membrane protein, the murinecytidine-5′-monophosphate-sialic acid transporter (CMP-Sia) (Newstead etal., Proc. Natl. Acad. Sci. USA 2007, 104, 13936-13941). The protein wasinitially extracted from S. cereviseae membranes with 1% DDM andisolated in buffer containing 0.03% DDM. The final purified protein (6mg/mL) was diluted 1:100 into solutions containing DDM or GDN at 0.042wt % (which corresponds to CMC+0.033 wt % for DDM and CMC+0.04 wt % forGDN). The DDM- and GDN-solubilized CMP-Sia were analyzed by gelfiltration before and after incubation for 2 hours at 30° C. (FIG. 5 a,b).

The results show GDN to be superior to DDM: CMP-Sia solubilized with DDMdisplays ˜50% integrity after the 2 hour period, while GDN-solubilizedprotein retains >90% integrity. The favorable effect of GDN on CMP-Siastability was further supported byN-[4-(7-diethylamino-4-methyl-3-coumarinyl) phenyl]maleimide (CPM) assayresults when the membrane proteins were evaluated at CMC+0.04 wt % (FIG.6 a) (Alexandrov et al., Structure 2008, 16, 351-359). Two othermembrane proteins were examined with the CPM assay, the rhomboidintramembrane serine protease GlpG (Urban, Biochem. J. 2010, 425,501-521) and succinate:quinone oxidoreductase (SQR) (Horsefield et al.,Curr. Protein Pept. Sci. 2004, 5, 107-118), both of which were expressedin Escherichia coli. In both cases, the results indicate that the newGLC/GDN amphiphiles are superior to DDM at maintaining native structure(FIGS. 6 b and 6 c).

The new amphiphiles were then evaluated for the ability to maintain theleucine transporter (LeuT) from Aquifex aeolicus in a functional state(Quick and Javitch, Proc. Natl. Acad. Sci. USA 2007, 104, 3603-3608).The transporter was initially extracted with DDM and then diluted withamphiphile-containing solutions to generate amphiphile concentration ofCMC+0.04 wt % or CMC+0.2 wt %. At both concentrations, GDN was veryeffective at maintaining LeuT activity, as indicated by binding ofradiolabeled leucine, with preservation of >95% of initial activityafter 12 days (FIG. 5 c). In contrast, DDM-solubilized LeuT lostsignificant activity over this period. The GLC amphiphiles, too, weresuperior to DDM in terms of maintaining LeuT activity, although they didnot match the effectiveness of GDN (FIG. 7).

To assess the new amphiphiles with a GPCR, a human β₂ adrenergicreceptor-T4-lysozyme fusion protein (β₂AR-T4L) was used (Rosenbaum etal., Science 2007, 318, 1266-1273). Stability was assessed via opticalabsorption measurements of β₂AR-T4L bound to the inverse agonistcarazolol. β₂AR-T4L was initially solubilized and purified with DDM, andthis detergent was then exchanged for the agent to be evaluated. Thefluorescence emission maximum of carazolol occurs at 356 nm in aqueoussolution, but emission is shifted to 341 nm in the receptor-bound state.The 341:356 nm peak intensity ratio was used to monitor the relativeamounts of intact and denatured β₂AR-T4L, with T_(m) defined as thetemperature at which the 341:356 nm peak intensity ratio is half-waybetween that of fully native receptor and the fully denatured receptor.FIG. 8 a shows how T_(m) varies as a function of amphiphileconcentration. At relatively low concentrations (<CMC+0.05 wt %), DDMwas superior to the new amphiphiles. However, GLC-2 and GDN becamesuperior to DDM at higher concentrations.

In the examples discussed above, conventional detergents such as DDMwere used to extract IMPs from the membrane, and then in most cases thesolution of detergent-solubilized protein was diluted withamphiphile-containing solutions to evaluate the new agents. With thisapproach it is possible that the small amount of residual conventionaldetergent could affect protein stability. To exclude this possibility,GLC-3 and GDN were used to extract wild type β₂AR (β₂AR WT) (Kobilka,Trends in Pharmacological Sciences 2011, 32, 213-218) from the membrane.Receptor activity was measured via a binding assay involving theantagonist [³H]-dihydroalprenolol. The DDM-solubilized receptor showedlow initial activity and rapidly decomposed (FIG. 8 b).GLC-3-solubilized receptor showed initial activity similar to that ofDDM, but in this case activity was maintained over 72 hours.GDN-solubilized β₂AR WT showed remarkable behavior: high initialactivity (>3-fold increase relative to that seen with DDM) that did notvary over 72 hours. GDN is therefore highly suitable for GPCRextraction.

We turned to a δ-opioid receptor-T4L fusion (δOR-T4L), another GPCR, tocompare GDN with the recently reported amphiphile MNG-3 (P. S. Chae, etal., Nat. Methods 2010, 7, 1003-1008), which has proven to be essentialfor crystallization of several other GPCR constructs (Rasmussen, et al.,Nature 2011, 469, 236-240; Rosenbaum, et al., Nature 2011, 469, 175-180;Rasmussen, et al., Nature 2011, 477, 540-541; Kruse, et al., Nature2012, 482, 552-556; Haga, et al., Nature 2012, 482, 547-551). Consistentwith prior observations, MNG-3-solubilized δOR-T4L showed higheractivity than DDM-solubilized δOR-T4L (FIG. 9). Remarkably,GDN-solubilized δOR-T4L displayed even higher activity. This dataindicates that GDN is significantly effective for GPCR solubilization.The ability of the new amphiphiles to stabilize membrane proteins thusallows for the ability to perform analyses and structural studies thatcould not be performed with other detergents such as DDM because harsherdetergents (e.g., DDM) can remove important lipid moieties from theproteins, causing them to denature and rapidly loose activity.

Because GDN displayed particularly favorable behavior in the precedingstudies, this agent was further characterized with melibiose permease(MelB), expressed in Salmonella typhimurium (Yousef and Guan, Proc.Natl. Acad. Sci. USA 2009, 106, 15291-15296). DDM (1.5 wt %) or GDN (1.5wt %) was used to extract MelB from S. typhimurium membranes at 0° C.for 10 min or 90 min and then aggregated material was removed viaultracentrifugation. The amount of MelB in solution was determined bySDS-PAGE with immunoblot detection (FIG. 10). DDM could quantitativelyextract MelB under these conditions; GDN was not quite as efficient inextraction, although a substantial yield of MelB was obtained.

The effect of DDM and GDN on MelB thermostability was assessed bysolubilizing the protein at the elevated temperatures for 90 min. DDMgave a high yield of soluble MelB at 45° C., but at 55° C. no solubleprotein was obtained. Presumably MelB denatured and aggregated at thehigher temperature in the presence of DDM. In contrast, GDN providedlarge amounts of soluble protein at 55° C. and even at 65° C.Interestingly, GDN could quantitatively extract the protein at elevatedtemperatures. This result indicates that GDN may be more useful forextracting membrane proteins at high temperatures relative to lowtemperatures (e.g., 4° C. or 25° C.). When MelB expressed in E. coli wasused, similar results were obtained (FIG. 11).

The favorable MelB extraction performance of GDN led to the examinationof this amphiphile for extraction of other IMPs. Comparable results wereobtained when the LHI-RC superassembly was extracted from R. capsulatusmembranes with either 2 wt % GDN or 1 wt % DDM (GDN molecular weight ismore than twice that of DDM) (FIG. 12 a). For β₂AR WT extraction frominsect cell membranes, 1 or 2 wt % GDN was more effective than was 1 wt% DDM; only a very small amount of β₂AR WT was detected with 1 wt % OG(FIG. 12 b). DDM and GDN were used to extract a CMP-Sia fusion proteinbearing green fluorescent protein (GFP) at the C-terminus, afterexpression in Saccharomyces cerevisiae; the amount of solubilizedprotein was estimated by total fluorescence. GDN (2 wt %), DDM (1 wt %)and OG (1 wt %) gave ˜70%, ˜80% and ˜50% extraction yields,respectively. Overall, results with several systems show that GDN isgenerally very effective at extracting embedded proteins from biologicalmembranes.

The results reported herein demonstrate that GDN is an extremely usefultool for membrane protein research. The GLC amphiphiles demonstrateuseful behavior for several types of IMPs. It is particularly noteworthythat the tests described herein include membrane protein systems thatvary in terms of structure and function. These studies have includedsystems, such as the R. capsulatus photosynthetic superassembly, LeuT,MelB and β₂AR, that display only limited stability when solubilized withconventional detergents. DDM is probably the most popular conventionaldetergent for IMP manipulations. The data described herein shows thatGDN consistently matches or exceeds DDM in terms of both extracting andstabilizing diverse membrane proteins.

The MNG amphiphile series was recently reported (Chae et al., Nat.Methods 2010, 7, 1003-1008). The MNG molecules are structurally quitedifferent from GDN and the MNG amphiphiles have already proven theirworth by enabling the acquisition of new GPCR crystal structures(Rasmussen et al., Nature 2011, 469, 236-240; Rasmussen et al., Nature2011, 469, 175-180; Rasmussen et al., Nature 2011, doi:10.1038/nature10361). Although this disclosure does not directly comparethe new steroidal agents with MNG amphiphiles, the fact that DDM wasused as a benchmark for both studies allows for the conclusion that GDNis generally comparable to the best MNG examples identified to date forIMP extraction and solubilization, based on results with multiple IMPsystems. Differences are evident in specific systems (e.g., GDN is a bitless effective than MNG amphiphiles in terms of β₂AR-T4Lthermostability, but GDN is superior in terms of MelB thermostability).Because the MNG and GDN molecular structures are very different, thesetwo types of amphiphile will manifest distinct and complementaryadvantages among the large set of membrane proteins that have yet to betamed in the laboratory.

Typical detergents such as DDM, OG and LDAO have simple alkyl chains asthe lipophilic groups. In the presence of a membrane protein, theseamphiphiles associate with one another to cover the hydrophobic surfacesof the protein, resulting in protein-detergent complexes (PDCs). Theoverall architectures of the amphiphiles introduced herein are neitherfacially amphiphilic nor polymeric. Consequently, the new agents areanticipated to associate with membrane protein similarly to classicaldetergents. Since, however, the lipophilic groups of the newsteroid-derived amphiphiles described herein are rigid and flat, thesemolecules will display a stronger tendency to associate withcomplementary protein surfaces than do conventional detergents, and thistendency underlies the favorable solubilization and stabilizationproperties documented herein.

Solubilization and Stability Assays

Light harvesting (LH) and reaction center (RC) complexes fromphotosynthetic bacteria (for example, R. capsulatus) are highly suitablefor use in solubilization assays. These complexes, normally embedded inthe bacterial membrane, are highly pigmented and several outcomes froman assay are possible, including no degradation, partial degradation orcomplete degradation upon solubilization, or no solubilization. Thus,graded comparative evaluations could be obtained for a set of candidatessuch as the carbohydrate-based amphiphiles described herein. In theengineered strain of R. capsulatus employed, the photosynthetic unit wascomprised of a very labile LHI complex and a more resilient RC complex.An ideal amphiphile will extract the intact LHI-RC superassembly from abacterial membrane preparation and maintain the natural interactionsamong the components. Amphiphiles with a more disruptive effect willdissociate and denature LHI, leaving only intact RC, and even harsheramphiphiles will cause RC degradation. Each of these various outcomescan be assessed unambiguously via optical spectroscopy.

Additional assays were carried out with reference to the followingprocedures. See also Example 3 below.

Bacteriorhodopsin Stability.

The procedure for the bR stability assay followed the protocol reportedby Bazzacco and coworkers (Biomacromolecules 2009, 10, 3317-3326).

R. capsulatus Superassembly Stability.

The stability of R. capsulatus superassembly was assessed according tothe protocol described by Chae and coworkers (J. Am. Chem. Soc. 2010,132, 16750-16752).

Thermal Stability Assay for CMP-Sia, GlpG, and SQR.

The thermal stability assays of these membrane proteins were performedas described by Chae and coworkers (J. Am. Chem. Soc. 2010, 132,16750-16752) using a temperature of 30° C. rather than 40° C.

CMP-Sia Gel Filtration.

Protein integrity was assessed using the procedure reported by Chae andcoworkers (Nat. Methods 2010, 7, 1003-1008) using CMP-Sia instead ofSQR.

LeuT Functional Assay.

LeuT functionality was measured according to the procedure reported byChae and coworkers (Nat. Methods 2010, 7, 1003-1008).

β₂AR-T4L Stability.

Stability of β₂AR-T4L was assessed by measuring the melting temperature(T_(m)) of the receptor according to the procedure reported by Chae andcoworkers (Nat. Methods 2010, 7, 1003-1008).

MelB Stability.

The protocol reported by Chae and coworkers (Nat. Methods 2010, 7,1003-1008) was used to evaluate MelB stability with DDM and GDN.

Solubilization of R. capsulatus Superassembly, β₂AR WT and CMP-SiaFusion Protein.

The procedure was performed according to the protocol reported by Chaeand coworkers (Nat. Methods 2010, 7, 1003-1008).

The amphiphiles were thus evaluated as membrane protein solubilizers andstabilizers and they compared very favorably with DDM, a standardreagent in the field. Compared to DDM, for example, one of the evaluatedamphiphiles forms micelles at one-tenth of the concentration requiredfor DDM. Compared with DDM, GDN performs similarly for extractingmembrane proteins from membranes, and stably retains the proteins in asoluble form within the micelles for substantially longer (>3 weeks)than DDM. Such improved stability (without sacrificing extractionefficiency) is a valuable trait for research tool amphiphiles.

The amphiphiles described herein require lower concentrations than manycommonly used surfactants to form stable micelles. Also, proteinsextracted with those amphiphiles demonstrate similar or higher activityand are extremely stable (i.e., remain soluble) in the micelle. In oneexample, the protein was stable in micelles of an amphiphile describedherein for >3 weeks, whereas the DDM stabilized protein activity beganto decay in a few hours or a few days.

The invention also provides a kit for enhancing the solubilization orstability of a proteinaceous macromolecule a biological sample, such asa membrane protein. The kit can include a solubilization reagent, suchas an amphiphile described herein, to solubilize at least one protein ina biological sample, one or more reagents such as buffers, enzymes,solvents, and/or other surfactants, and optionally directions for thesolubilization and/or recover a protein in a biological sample, and/ordirections to isolate and/or resolve a protein in a biological sample.

The following Examples are intended to illustrate the above inventionand should not be construed as to narrow its scope. One skilled in theart will readily recognize that the Examples suggest many other ways inwhich the invention could be practiced. It should be understood thatnumerous variations and modifications may be made while remaining withinthe scope of the invention.

EXAMPLES Example 1 Preparation of GLC Amphiphiles

A. Synthesis of Perbenzoylated Maltosylbromide.

This compound was prepared by following the reported protocol forperbenzoylated lactosylbromide (Kamath et al. Carbohydr. Res. 2004, 339,1141-1146) with modifications as follows. To a solution of maltosemonohydrate (30 g, 0.083 mol) in pyridine (300 mL) was added slowlybenzoyl chloride (106 mL, 0.92 mol) and a catalytic amount (˜0.2 g) ofdimethylamino-pyridine (DMAP) at 0° C. The resulting solution wasallowed to warm to RT and stirred for 20 hours at the same temperature.The solution was taken up with EtOAc (300 mL) and was washed with aniced aqueous 2N HCl solution until the aqueous phase became acidic. Theneutralized organic layer was washed with brine (2×200 mL). Thecollected organic layer was dried over anhydrous Na₂SO₄ and removed byrotary evaporation to give crude syrup. This crude syrup was used forthe next reaction without further purification. The crude material wasdissolved in dried CH₂Cl₂ (100 mL) and to the solution was added 33 wt %HBr-acetic acid (100 mL) at 0° C. under N₂ conditions. The mixture wasstirred at 0° C. for 4 hr. The solution was washed with iced water andsaturated NaHCO₃ solution until the aqueous layer became slightly basic.The neutralized organic solution was washed with brine, dried overanhydrous Na₂SO₄ and removed by rotary evaporation to make crude syrup.The syrup was dissolved in ether (˜500 mL) and stored at RT until whiteprecipitates was formed. The white precipitates were collected on theglass filter and washed with ether three times. The filtered solid wasdried in vacuo to afford perbenzoylated maltosylbromide as a white solid(80 g, 80% in two steps). This product was used for the next reactionwithout further purification. ¹H NMR (300 MHz, CDCl₃): δ 8.13-8.06 (m,2H), 8.02-7.96 (m, 2H), 7.91-7.84 (m, 4H), 7.77-7.64 (m, 4H), 7.69-7.63(m, 2H), 7.63-7.15 (m, 21H), 6.76 (d, J=3.7 Hz, 2H), 6.16 (t, J=9.4 Hz,2H), 6.10 (t, J=9.2 Hz, 1H), 5.79 (d, J=4.0 Hz, 1H), 5.68 (t, J=9.6 Hz,1H), 5.28 (dd, J=10.8, 4.0 Hz, 1H), 5.09 (dd, J=10.0, 3.8 Hz, 1H),4.96-4.87 (m, 1H), 4.84-4.75 (m, 1H), 4.72-4.62 (m, 3H), 4.59-4.39 (m,1H).

B. General Procedure for Glycosylation Reactions.

This reaction was performed according to a literature method (Ashton etal., Chem. Eur. J. 1996, 2, 1115-1128) with slight modification. Amixture of hydroxyl-containing compound (having two hydroxyl groups),AgOTf (2.4 equiv.), and 2,4,6-collidine (1.8 equiv.) in anhydrous CH₂Cl₂(40 mL) was stirred at −45° C. A solution of perbenzoylatedmaltosylbromide (2.4 equiv.) in CH₂Cl₂ (40 mL) was added dropwise over0.5 hours to this suspension. Stirring was continued for 0.5 hours at−45° C., after which the reaction mixture was allowed to warm to 0° C.and was left stifling for 1.5 hours. After completion of reaction (asdetected by TLC), pyridine was added and the reaction mixture wasdiluted with CH₂Cl₂ (40 mL) before being filtered over celite. Thefiltrate was washed successively with a 1 M aqueous Na₂S₂O₃ solution (40mL), a 0.1 M aqueous HCl solution (40 mL), and brine (2×40 mL). Theorganic layer was then dried with anhydrous Na₂SO₄ and the solvents wereremoved by rotary evaporation. The residue was purified by silica gelcolumn chromatography (EtOAc/hexane) providing desired product as aglassy solid.

C. General Procedure for De-O-Benzoylations Under Zemplén's Conditions.

The O-benzoylated compounds were dissolved in MeOH and then treated withthe required amount of a methanolic solution of 0.5 M NaOMe such thatthe final concentration of NaOMe was 0.05 M. The reaction mixture wasleft stirring for 6 hours at room temperature, and then neutralized withAmberlite IR-120 (H⁺ form) resin. The resin was removed by filtrationand washed with MeOH and solvent was removed from the combined filtratein vacuo. The residue was purified by silica gel column chromatography(MeOH/CH₂Cl₂). Further purification carried out by recrystallizationusing CH₂Cl₂/MeOH/diethyl ether afforded the fully de-O-benzoylatedproduct as a white solid.

D. Synthesis and Characterization of GLC Amphiphiles.

Preparation of new amphiphiles GLC-1, GLC-2, and GLC-3 is illustrated inthe synthetic schemes of FIGS. 13 and 14.

Compound 1 was synthesized by a modified literature protocol (Taotafa etal., Org. Lett. 2000, 2, 4117-4120). ¹H NMR (300 MHz, CDCl₃): δ 3.26 (s,3H), 3.24-3.08 (m, 1H), 2.46-2.32 (m, 1H), 2.32-2.16 (m, 1H), 1.96-1.50(m, 10H), 1.50-0.94 (m, 18H), 0.94-0.82 (m, 6H), 0.64 (s, 3H); ¹³C NMR(75 MHz, CDCl₃): δ 80.7, 56.7, 56.2, 55.7, 43.0, 42.3, 40.6, 40.4, 36.1,35.6, 35.5, 35.1, 32.9, 31.2, 31.0, 28.4, 27.6, 27.0, 26.6, 24.4, 23.6,21.0, 18.5, 12.3; MS (MALDI-TOF): calcd. for C₂₅H₄₂O₃ [M+Na]⁺ 413.3027.found 413.3017.

Compound 2. Methylated lithocholic acid (1) (1.5 g, 3.8 mmol), serinol(0.41 g, 4.6 mmol), 1-hydroxybenzotriazole monohydrate (HOBt) (0.61 g,4.6 mmol) was dissolved in anhydrous DMF (30 mL).1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl)(0.87 g, 4.55 mmol) was added in small portions at 0° C. and theresulting solution left stirring at room temperature for 20 h. Thesolution was taken up with EtOAc (100 mL) and was washed successivelywith a 1 M aqueous NaHCO₃ solution (100 mL), a 0.1 M aqueous HClsolution (100 mL) and brine (2×100 mL). Then the organic layer was driedwith anhydrous Na₂SO₄ and the solvent was removed by rotary evaporation.The reaction mixture was precipitated with ether (100 mL) and theresulting solid was collected and dried in vacuo to affordamide-containing diol (2) as a white solid (1.60 g, 91%). This productwas used for next reaction without further purification. ¹H NMR (300MHz, CDCl₃): δ 6.86 (d, J=7.9 Hz, 1H), 3.88-3.80 (m, 4H), 3.76-3.64 (m,2H), 3.64-3.52 (m, 2H), 3.36 (s, 3H), 3.26-3.12 (m, 1H), 2.34-2.21 (m,1H), 2.17-2.05 (m, 1H), 1.98-1.50 (m, 9H), 1.48-0.94 (m, 16H), 0.94-0.84(m, 6H), 0.65 (s, 3H); ¹³C NMR (75 MHz, CDCl₃): δ 175.4, 80.7, 61.4,56.5, 56.0, 55.4, 52.5, 49.3, 49.0, 48.7, 48.4, 42.8, 42.1, 40.4, 40.2,35.9, 35.6, 35.2, 34.9, 32.7, 31.8, 28.2, 27.3, 26.7, 26.4, 24.2, 23.4,20.8, 18.3, 12.0; MS (MALDI-TOF): calcd. for C₂₈H₄₉NO₄ [M+Na]⁺ 486.3554.found 486.3570.

Compound 3. LiAlH₄ (0.44 g, 1.5 mmol) was added slowly to compound 1(1.5 g, 3.8 mmol) dissolved in THF (50 mL) at 0° C. The mixture wasstirred at RT for 1 day, quenched with MeOH, water, a 1 N aqueous HClsolution successively at 0° C. and then extracted with diethyl ether(2×50 mL). The combined organic layer was washed with brine and driedwith anhydrous Na₂SO₄. The residue was purified by silica gel columnchromatography (EtOAc/hexane) providing a desired product (3) as a whitesolid (1.3 g, 89%). ¹H NMR (300 MHz, CDCl₃): δ 3.60 (t, J=6.0 Hz, 2H),3.34 (s, 3H), 3.21-3.11 (m, 1H), 2.01-1.51 (m, 10H), 1.50-0.96 (m, 18H),0.96-0.82 (m, 6H), 0.64 (m, 3H); ¹³C NMR (75 MHz, CDCl₃): δ 80.6, 63.7,56.7, 56.4, 55.7, 42.9, 42.2, 40.5, 40.4, 36.0 35.8, 35.5, 35.1, 32.9,32.0, 29.6, 28.5, 27.5, 27.0, 26.6, 24.4, 23.6, 21.0, 18.8, 12.2; MS(MALDI-TOF): calcd. for C₂₅H₄₄O₂ [M+NH₄]⁺ 394.3680. found 394.3683.

Compound 4. To a solution of alcohol (3) (0.88 g, 2.3 mmol) and carbontetrabromide (0.79 g, 3.0 mmol) in CH₂Cl₂ (100 mL) was addedtriphenylphosphine (Ph₃P) at 0° C. The solution was stirred at 0° C. for1 hr and then continued the stirring at RT for 15 hr. The solvent wasevaporated and then the 1:15 mixture of CH₂Cl₂ and hexane (100 mL) wasadded to the residue to precipitate out the oxidized side product oftriphenylphosphine. After filtration and evaporation, the residue waspurified by silica gel column chromatography (EtOAc/hexane) providing adesired product (4) as a white solid (0.92 g, 90%). ¹H NMR (300 MHz,CD₃OD): δ 3.43-3.29 (m, 5H), 3.21-3.09 (m, 1H), 2.01-1.47 (m, 11H),1.47-0.99 (m, 17H), 0.99-0.84 (m, 7H), 0.64 (s, 3H); ¹³C NMR (75 MHz,CD₃OD): δ 80.6, 56.8, 56.7, 56.3, 55.8, 42.9, 42.3, 40.4, 36.1, 35.5,35.4, 35.1, 34.8, 34.7, 33.0, 29.9, 28.5, 27.6, 27.0, 26.6, 24.4, 23.6,21.0, 18.9, 12.3; MS (MALDI-TOF): calcd. for C₂₅H₄₃O₂Br [M+NH₄]⁺456.2836. found 456.2118.

Compound 5. 1,1,1-Tris(hydroxymethyl)ethane (1.3 g, 11.2 mmol) isdissolved in 40 mL of DMF and NaH (0.45 g, 11.2 mmol) was added. Bromide(4) (1.6 g, 3.7 mmol) was added to this solution and the mixture wasstirred for 2 hr at 60° C. After adding water (100 mL), the resultingresidue was extracted with ether (2×100 mL). The combined organic layerwas washed with brine and dried with anhydrous Na₂SO₄. The residue waspurified by silica gel column chromatography (EtOAc/hexane) providingalkyl-containing diol (5) as a white solid (1.1 g, 60%). ¹H NMR (300MHz, CD₃OD): δ 3.69 (d, J=11.0 Hz, 2H), 3.56 (d, J=11.0 Hz, 2H),3.46-3.34 (m, 7H), 3.21-3.11 (m, 1H), 3.01-2.79 (br s, 2H), 2.01-1.57(m, 9H), 1.57-0.92 (m, 20H), 0.92-0.71 (m, 11H), 0.63 (s, 3H); ¹³C NMR(75 MHz, CD₃OD): δ 80.6, 77.0, 72.5, 67.9, 56.6, 56.2, 56.2, 55.6, 42.9,42.8, 42.2, 40.9, 40.8, 40.5, 40.3, 36.0, 35.7, 35.4, 35.1, 35.0, 32.3,28.4, 27.4, 26.9, 26.5, 26.2, 24.3, 23.5, 20.9, 18.8, 18.6, 17.3, 12.2;MS (MALDI-TOF): calcd. for C₃₀H₅₄O₄ [M+H]⁺ 479.4095. found 479.4096.

Compound 6. To a solution of bromide (4; 0.92 g, 2.1 mmol) and diethylmalonate (1.6 g, 10.4 mmol) in 1:1 mixture of THF and DMF (80 mL) wasadded K₂CO₃ (1.5 g, 10.5 mmol). The mixture was heated at 90° C. for 15hr, quenched with water (100 mL) at 0° C. and then extracted withdiethyl ether (2×100 mL). The combined organic layer was washed withbrine and dried with anhydrous Na₂SO₄. The crude product was used forthe next reaction without further purification. The crude product wasdissolved in THF (50 mL) and LiAlH₄ (0.52 g, 14.0 mmol) was added slowlyto the solution at 0° C. The mixture was stirred at RT for 1 day,quenched with MeOH, water, a 1 N aqueous HCl solution successively at 0°C. and then extracted with diethyl ether (2×50 mL). The combined organiclayer was washed with brine and dried with anhydrous Na₂SO₄. The residuewas purified by silica gel column chromatography (EtOAc/hexane)providing alkyl-containing diol (6) as a white solid (0.85 g, 93% (twosteps)). ¹H NMR (300 MHz, CD₃OD): 3.89-3.81 (m, 2H), 3.67-3.59 (m, 2H),3.35 (s, 3H), 3.24-3.10 (m, 1H), 3.54 (br s, 2H), 2.02-1.48 (m, 10H),1.48-0.92 (m, 25H), 0.92-0.71 (m, 10H), 0.63 (s, 3H); ¹³C NMR (75 MHz,CD₃OD): δ 80.7, 67.1, 66.8, 56.7, 56.5, 55.7, 42.9, 42.3, 40.6, 40.4,36.4, 36.1, 35.9, 35.5, 35.1, 33.0, 28.6, 28.4, 27.5, 27.0, 26.6, 24.4,24.0, 23.6, 21.0, 18.8, 12.2; MS (MALDI-TOF): calcd. for C₂₈H₅₀O₃[M+NH₄]⁺ 452.4099. found 452.4102.

GLC-1a was synthesized according to the general procedure forglycosylation. ¹H NMR (300 MHz, CD₃OD): 5.77 (d, J=8.1 Hz, 1H),5.44-5.19 (m, 5H), 5.06 (dt, J=10.2, 2.0 Hz, 2H), 4.90-4.75 (m, 4H),4.55-4.43 (m, 4H), 4.32-4.15 (m, 5H), 4.09-3.93 (m, 6H), 3.80-3.67 (m,4H), 3.56-3.46 (m, 1H), 3.35 (m, 3H), 3.23-3.09 (m, 1h), 2.16 (s, 6H),2.11 (s, 6H), 2.08-1.93 (m, 32H), 1.93-0.95 (m, 29H), 0.95-0.80 (m, 8H),0.63 (s, 3H); ¹³C NMR (75 MHz, CD₃OD): δ 173.5, 170.7, 170.6, 170.5,170.3, 170.2, 170.1, 169.9, 169.8, 169.6, 101.0, 100.8, 95.8, 80.6,75.4, 75.2, 73.0, 72.9, 72.6, 72.5, 72.4, 72.2, 70.2, 69.5, 68.7, 68.6,68.2, 62.9, 61.6, 56.2, 55.7, 42.9, 42.2, 40.5, 40.4, 36.0, 35.7, 35.0,33.5, 33.0, 31.7, 28.4, 27.5, 27.0, 24.4, 23.6, 21.1, 21.0, 20.9, 20.8,18.6, 12.2; MS (MALDI-TOF): calcd. for C₁₅₀H₁₄₅NO₃₈ [M+Na]⁺ 2590.9.found 2591.4.

GLC-2a was synthesized according to the general procedure forglycosylation. ¹H NMR (300 MHz, CD₃OD): 8.12-8.02 (m, 4H), 8.02-7.95 (m,7H), 7.95-7.88 (m, 4H), 7.88-7.83 (m, 4H), 7.83-7.77 (m, 4H), 7.77-7.67(m, 4H), 7.67-7.16 (m, 45H), 6.13 (t, J=10.0 Hz, 2H), 5.72-5.60 (m, 4H),5.39 (t, J=9.5 Hz, 2H), 5.20-5.08 (m, 4H), 4.70-4.43 (m, 4H), 4.40-4.16(m, 8H), 3.56-3.37 (m, 5H), 3.34 (s, 3H), 3.26-3.12 (m, 2H), 3.06 (q,J=10.1 Hz, 2H), 2.98-2.89 (m, 3H), 2.84 (d, J=9.2 Hz, 1H), 1.95-0.95 (m,34H), 0.95-0.82 (m, 6H), 0.82-0.73 (m, 6H), 0.59 (s, 3H); ¹³C NMR (75MHz, CD₃OD): δ 166.3, 166.2, 166.0, 165.7, 165.2, 165.0, 133.9, 133.7,133.6, 133.4, 133.3, 133.2, 130.1, 129.9, 129.8, 129.6, 129.3, 129.2,129.1, 129.0, 128.9, 128.8, 128.6, 128.5, 128.4, 128.3, 101.1, 101.0,95.9, 80.6, 74.8, 73.6, 72.5, 72.4, 72.3, 71.4, 70.0, 69.2, 69.1, 66.0,63.5, 62.7, 56.6, 56.3, 55.7, 42.8, 42.2, 40.5, 40.4, 40.3, 36.0, 35.6,35.5, 35.1, 33.0, 32.2, 28.4, 27.5, 27.0, 26.6, 26.2, 24.4, 23.6, 21.0,18.8, 17.2, 15.4, 14.4, 12.2; MS (MALDI-TOF): calcd. for C₁₅₂H₁₅₀O₃₈[M+Na]⁺ 2606.0. found 2606.5.

GLC-3a was synthesized according to the general procedure forglycosylation. ¹H NMR (300 MHz, CD₃OD): 8.10-7.90 (m, 15H), 7.89-7.83(m, 4H), 7.83-7.76 (m, 4H), 7.76-7.68 (m, 4H), 7.68-7.13 (m, 42H), 6.13(t, J=10.0 Hz, 2H), 5.74-5.59 (m, 4H), 5.41-5.30 (m, 2H), 5.21-5.06 (m,4H), 4.72-4.48 (m, 4H), 4.41-4.16 (m, 8H), 3.69-3.56 (m, 2H), 3.34 (s,3H), 3.34-3.25 (m, 2H), 3.21-3.10 (m, 1H), 3.10-2.92 (m, 2H), 2.79 (t,J=9.8 Hz, 1H), 1.92-1.44 (m, 11H), 1.44-1.09 (m, 15H), 1.09-0.79 (m,14H), 0.79-0.69 (m, 3H), 0.56 (s, 3H) ¹³C NMR (75 MHz, CD₃OD): δ 166.3,166.0, 165.7, 165.2, 165.1, 165.0, 133.7, 133.6, 133.4, 133.3, 130.2,130.1, 130.0, 129.8, 129.7, 129.6, 129.5, 129.4, 129.2, 129.1, 129.0,128.6, 128.4, 101.2, 95.8, 80.6, 77.4, 74.8, 74.7, 72.2, 71.4, 70.0,69.1, 62.7, 60.6, 56.6, 55.7, 42.8, 42.3, 40.5, 40.3, 36.0, 35.9, 35.5,35.1, 33.0, 28.5, 28.2, 27.5, 27.0, 24.4, 23.9, 23.6, 21.2, 21.0, 18.7,14.4, 12.2; MS (MALDI-TOF): calcd. for C₁₅₀H₁₄₆O₃₇ [M+Na]⁺ 2561.9. found2562.5.

GLC-1 was synthesized according to the general procedure forde-O-benzoylation. ¹H NMR (300 MHz, CDCl₃): δ 5.15 (d, J=3.6 Hz, 2H),4.33 (t, J=7.0 Hz, 2H), 4.01-3.74 (m, 9H), 3.73-3.15 (m, 25H), 2.36-2.20(m, 1H), 2.20-2.06 (m, 1H), 2.06-1.52 (m, 10H), 1.52-1.02 (m, 18H),1.02-0.88 (m, 7H), 0.69 (s, 3H); ¹³C NMR (75 MHz, CDCl₃): δ 176.9,103.0, 82.1, 81.4, 77.8, 76.7, 75.2, 74.9, 74.8, 74.7, 74.2, 71.6, 62.8,58.0, 57.5, 56.0, 44.0, 43.5, 42.0, 37.3, 37.0, 36.1, 28.5, 27.8, 24.1,22.1, 19.1, 12.8; MS (MALDI-TOF): calcd. for C₅₂H₈₉O₂₄ [M+Na]⁺1134.5667.found 1134.5703.

GLC-2 was synthesized according to the general procedure forde-O-benzoylation. ¹H NMR (300 MHz, CDCl₃): δ 5.19 (d, J=3.6 Hz, 2H),4.35 (t, J=7.7 Hz, 2H), 4.00-3.77 (m, 8H), 3.77-3.51 (m, 12H), 3.51-3.14(m, 15H), 2.11-1.05 (m, 26H), 1.05-0.91 (m, 9H), 0.72 (s, 3H); ¹³C NMR(75 MHz, CDCl₃): δ 105.1, 103.0, 82.2, 81.5, 78.0, 76.7, 75.2, 74.9,74.3, 73.6, 73.4, 73.2, 71.6, 62.9, 62.3, 58.0, 57.8, 56.0, 44.0, 43.5,42.1, 42.0, 41.7, 37.4, 37.0, 36.4, 36.1, 34.0, 33.7, 29.6, 28.5, 27.9,27.8, 27.5, 25.4, 24.1, 22.1, 19.4, 18.0, 12.7; MS (MALDI-TOF): calcd.for C₅₄H₉₄O₂₄ [M+Na]⁺ 1149.6027. found 1149.6029.

GLC-3 was synthesized according to the general procedure forde-O-benzoylation. ¹H NMR (300 MHz, CDCl₃): δ 5.16 (d, J=3.6 Hz, 2H),4.32 (t, J=7.7 Hz, 2H), 3.99-3.74 (m, 8H), 3.74-3.55 (m, 10H), 3.55-3.15(m, 12H), 2.10-1.02 (m, 31H), 1.02-0.83 (m, 7H), 0.68 (s, 3H); ¹³C NMR(75 MHz, CDCl₃): δ 104.9, 104.8, 103.0, 82.2, 81.5, 78.0, 76.7, 75.2,74.9, 74.3, 71.6, 71.4, 62.9, 62.4, 58.0, 57.9, 56.0, 44.0, 43.6, 42.0,41.7, 40.9, 37.6, 37.4, 37.2, 36.4, 36.1, 34.0, 30.0, 29.6, 28.5, 28.0,27.8, 25.4, 24.8, 24.1, 22.1, 19.4, 12.7; MS (MALDI-TOF): calcd. forC₅₂H₉₀O₂₃ [M+Na]⁺ 1105.5766. found 1105.5719.

Example 2 Preparation of GDN Amphiphiles

The synthetic scheme for the preparation of amphiphile GDN isillustrated in FIG. 15.

Compound 7. Ethyl diazoacetate (1.8 g, 15.7 mmol) was added to asolution of diosgenin (5.0 g, 12.1 mmol) dissolved in anhydrous CH₂Cl₂(100 mL) under N₂ atmosphere. BF₃.etherate (0.083 g, 0.67 mmol) was thenadded to the solution and then the resulting reaction mixture at RT for1.5 days. The reaction mixture was quenched with a saturated aqueousNaHCO₃ solution and extracted with ethyl acetate (200 mL). The organiclayer was washed with water (200 mL) and dried with anhydrous Na₂SO₄.The residue was purified by silica gel column chromatography(EtOAc/hexane) providing a desired product (7) as a white solid (3.3 g,55%). ¹H NMR (300 MHz, CDCl₃): δ 5.35 (d, J=5.2 Hz, 1H), 4.41 (q, J=7.3Hz, 1H), 4.20 (q, J=7.0 Hz, 2H), 4.12 (s, 2H), 3.52-3.42 (m, 1H), 3.37(t, J=10.6 Hz, 1H), 3.28-3.18 (m, 1H), 2.46-2.34 (m, 1H), 2.34-2.18 (m,1H), 2.07-1.80 (m, 5H), 1.80-1.34 (m, 13H), 1.28 (t, J=7.2 Hz, 3H),1.30-1.04 (m, 3H), 1.04-0.89 (m, 8H), 0.89-0.72 (m, 6H); ¹³C NMR (75MHz, CDCl₃): δ 171.1, 140.8, 124.0, 121.9, 109.5, 81.0, 80.2, 67.0,66.0, 62.3, 61.0, 56.7, 50.3, 41.8, 40.5, 40.0, 38.9, 37.3, 37.2, 32.3,32.1, 31.6, 30.5, 29.0, 28.3, 21.0, 19.6, 17.3, 16.5, 14.7, 14.4; MS(MALDI-TOF): calcd. for C₃₁H₄₈O₅ [M+NH₄]⁺ 518.3840. found 518.3837.Compound 8. This compound was synthesized via the synthetic protocol ofcompound 3 by using compound 7 as a starting material. Yield: 89%; ¹HNMR (300 MHz, CDCl₃): δ 5.35 (d, J=5.2 Hz, 1H), 4.41 (q, J=7.3 Hz, 1H),3.72 (t, J=4.6 Hz, 2H), 3.59 (t, J=4.6 Hz, 2H), 3.52-3.42 (m, 1H), 3.37(t, J=10.6 Hz, 1H), 3.26-3.11 (m, 1H), 2.45-2.31 (m, 1H), 2.26-2.14 (m,1H), 2.08-1.82 (m, 6H), 1.82-1.37 (m, 12H), 1.37-1.05 (m, 4H), 1.05-0.88(m, 8H), 0.85-0.69 (m, 6H); ¹³C NMR (75 MHz, CDCl₃): δ 141.0, 121.7,109.5, 81.0, 79.6, 69.2, 68.3, 67.0, 62.3, 56.7, 50.3, 41.8, 40.5, 40.0,37.4, 37.2, 32.3, 32.1, 31.6, 30.5, 29.0, 28.6, 21.1, 19.6, 17.3, 16.5,14.7; MS (MALDI-TOF): calcd. for C₂₉H₄₆O₄ [M+NH₄]⁺ 476.3735. found476.3739.

Compound 9. This compound was synthesized via the synthetic protocol ofcompound 4 by using compound 8 as a starting material. Yield: 86%; ¹HNMR (300 MHz, CDCl₃): δ 5.35 (s, 1H), 4.41 (q, J=7.3 Hz, 1H), 3.78 (t,J=6.8 Hz, 2H), 3.52-3.32 (m, 4H), 3.27-3.15 (m, 1H), 2.42-2.31 (m, 1H),2.29-2.16 (m, 1H), 2.08-1.81 (m, 5H), 1.81-1.37 (m, 12H), 1.36-1.05 (m,4H), 1.05-0.92 (7H), 0.92-0.73 (m, 7H); ¹³C NMR (75 MHz, CDCl₃): δ140.9, 121.8, 109.5, 81.0, 79.8, 68.3, 67.1, 62.3, 56.7, 50.3, 41.8,40.5, 40.0, 39.3, 37.4, 37.2, 32.3, 32.1, 31.6, 31.1, 30.5, 29.0, 28.6,21.1, 19.6, 17.4, 16.5, 14.7; MS (MALDI-TOF): calcd. forC₂₉H₄₅O₃Br[M+H]⁺ 521.2625. found 521.2621.

Compound 10. This compound was synthesized via the synthetic protocol ofcompound 6 by using compound 9 as a starting material. Yield (twosteps): 90%; ¹H NMR (300 MHz, CDCl₃): δ 5.34 (d, =5.2 Hz, 1H), 4.41 (q,=7.4 Hz, 1H), 3.78-3.63 (m, 4H), 3.57 (t, =5.6 Hz, 2H), 3.52-3.42 (m,1H), 3.37 (t, =10.6 Hz, 1H), 3.27-3.08 (m, 1H), 2.87 (s, 2h), 2.45-2.30(m, 1H), 2.27-2.12 (m, 1H), 2.08-1.34 (m, 21H), 1.34-1.05 (m, 4H),1.05-0.88 (m, 8H), 0.88-0.72 (m, 7H); ¹³C NMR (75 MHz, CDCl₃): δ 140.8,121.8, 109.5, 81.0, 79.6, 67.1, 66.4, 65.4, 62.3, 56.7, 41.8, 50.3,40.5, 40.1, 39.1, 37.3, 37.2, 32.3, 32.0, 31.6, 30.5, 29.5, 29.0, 28.5,21.1, 19.6, 17.3, 16.5, 14.7; MS (MALDI-TOF): calcd. for C₃₂H₅₂O₅[M+Na]⁺539.3707. found 539.3714.

GDNa was synthesized according to the general procedure forglycosylation. ¹H NMR (300 MHz, CDCl₃): δ 8.14-7.90 (m, 15H), 7.90-7.83(m, 4H), 7.83-7.77 (m, 4H), 7.77-7.68 (m, 4H), 7.68-7.15 (m, 42H), 6.13(t, J=10.0 Hz, 2H), 5.73-5.59 (m, 4H), 5.35 (q, J=9.7 Hz, 2H), 5.29-5.03(m, 8H), 4.71-4.48 (m, 4H), 4.48-4.14 (m, 9H), 3.69-3.57 (m, 2H),3.53-3.23 (m, 7H), 3.13-2.92 (m, 4H), 2.85-2.74 (m, 1H), 2.32-2.20 (m,1H), 2.20-1.81 (m, 5H), 1.80-1.70 (m, 5H), 1.70-1.55 (m, 4H), 1.55-1.40(m, 4H), 1.40-1.02 (m, 8H), 1.02-0.94 (m, 3H), 0.94-0.83 (m, 6H),0.83-0.70 (m, 5H); ¹³C NMR (75 MHz, CDCl₃): δ 166.3, 166.2, 166.0,165.6, 165.2, 165.1, 165.0, 141.1, 133.7, 133.6, 133.4, 133.3, 130.1,129.9, 129.8, 129.6, 129.5, 129.3, 129.1, 129.0, 128.9, 128.8, 128.7,128.6, 128.4, 128.3, 121.3, 109.3, 101.1, 95.8, 81.0, 78.9, 74.7, 72.3,72.2, 70.0, 69.2, 69.1, 67.0, 62.7, 60.6, 56.7, 50.2, 41.8, 40.4, 40.0,37.3, 37.1, 32.2, 32.0, 31.6, 30.5, 29.0, 28.5, 21.0, 19.5, 17.3, 16.5,15.5, 14.7; MS (MALDI-TOF): calcd. for C₁₅₄H₁₄₈O₃₉ [M+Na]⁺ 2643.9. found2644.6.

GDN was synthesized according to the general procedure forde-O-benzoylation. ¹H NMR (300 MHz, CDCl₃): δ 5.37 (d, J=5.2 Hz, 1H),5.15 (d, J=3.4 Hz, 2H), 4.39 (q, J=7.7 Hz, 1H), 4.10 (d, J=7.6 Hz, 2H)3.98-3.74 (m, 8H), 3.72-3.54 (m, 12H), 3.54-3.47 (m, 3H), 3.47-3.40 (m,3H), 3.40-3.32 (m, 2H), 3.32-3.08 (m, 5H), 2.43-2.30 (m, 1H), 2.20-1.82(m, 3H), 1.82-1.06 (m, 18H), 1.06-1.00 (m, 4H), 0.96 (d, J=6.9 Hz, 4H),0.85-0.74 (m, 6H) ¹³C NMR (75 MHz, CDCl₃): δ 142.2, 122.6, 110.7, 104.9,104.7, 103.1, 82.4, 81.5, 80.6, 78.0, 76.7, 75.2, 75.0, 74.3, 71.7,70.8, 68.0, 67.2, 63.9, 62.9, 62.4, 58.0, 51.8, 43.1, 41.6, 41.8, 40.4,38.6, 38.3, 38.0, 33.3, 33.0, 32.9, 32.6, 31.6, 30.0, 29.9, 29.7, 22.2,20.0, 17.7, 16.9, 15.1; MS (MALDI-TOF): calcd. for C₅₆H₉₂O₂₅ [M+Na]⁺1187.5820. found 1187.5769.

Example 3 Protein Stability Evaluation

Bacteriorhodopsin Stability.

The procedure for the bR stability assay generally followed the reportedprotocol (Bazzacco et al., Biomacromolecules 2009, 10, 3317-3326).Frozen aliquots of purple membranes containing bR at 184 μM were thawedat room temperature and solubilized by using an octylthioglucoside (OTG)solution for 24 hr at 4° C. in a dark room. For this purpose, OTG(CMC=0.28 wt %) was used at 2.0 wt % in 10 mM sodium phosphate (pH 6.9).Membrane debris was then removed from the solubilized material viaultracentrifugation at 200,000 g at 4° C. for 20 min. The supernatant,including bR protein, was transferred into individual DDM, GLC, or GDNsolutions, giving final concentration of OTG: new amphiphiles=0.2 wt%:0.8 wt % (1:4) or 0.2 wt %:1.6 wt % (1:8). The stability of bR in eachsolution was monitored by measuring absorbance at 554 nm over 20 days.

Solubilization and Stability Assay for R. capsulatus Superassembly.

The solubilization and stability of the R. capsulatus superassembly wereassessed according to the published protocol (Chae et al., J. Am. Chem.Soc. 2010, 132, 16750-16752). Briefly, specialized photosyntheticmembranes obtained from an engineered strain of Rhodobacter (R.)capsulatus, U43[pUHTM86Bgl], lacking the LHII light-harvesting complex,were used. Solubilization of the LHI-RC superassembly began by thawing,homogenizing, and incubating frozen aliquots of R. capsulatus membranesat 32° C. for 30 min. Subsequent 30-min incubation was performed afteradding DDM or LDAO at 1.0 wt % or GDN at 2.0 wt % in the solid form. Thesolution was then subjected to ultracentrifugation at 315,000 g at 4° C.for 30 min to remove membrane debris. To assess solubilizationefficiency, UV-Vis spectra of the solubilized protein solutions weremeasured in a range of 650˜950 nm.

For the stability assay, DDM-solubilized material was transferred into anew microcentrifuge tube containing Ni-NTA resin (Qiagen, Inc.;Valencia, Calif.; pre-equilibrated and stored in an equal volume ofbuffer containing 10 mM Tris, pH 7.8, and 100 mM NaCl). After a 1 hincubation at 4° C. for binding, the resins were washed twice with 0.5mL of binding buffer (a pH 7.8 Tris solution containing DDM at 1×CMC)and eluted three times with 0.20 mL elution buffer aliquots containing 1M imidazole (otherwise, this buffer was identical to binding buffer; thepH of each solution was readjusted to pH=7.8). The DDM-purifiedsolutions were collected and diluted with 0.4 mL of the binding buffer.Then small aliquots (0.05 mL) of the DDM-purified protein solutions weremixed with 0.95 mL GLC or GDN solutions at concentrations CMC+0.04 wt %,CMC+0.2 wt % or CMC+1.0 wt %. UV-Vis spectra of these solutions weremonitored at room temperature over 20 days. Protein degradation wasassessed by measuring the 875 nm/680 nm absorbance ratio.

Membrane Solubilization and Protein Purification (CMP-Sia, GlpG andSQR).

CMP-Sia and GlpG were expressed as fusion proteins with a C-terminalGFP-His tag in in Saccharomyces cerevisiae and Escherichia colirespectively. All steps were carried out at 4° C. Membranes containingCMP-Sia and GlpG were resuspended in PBS, 10 mM imidazole pH 8.0, 150 mMNaCl, 10% glycerol and solubilized in 1% DDM for 1 hour with mildagitation. Supernatant containing DDM-solubilized protein was harvestedafter ultracentrifugation at 100,000 g for 45 mM. The GFP-His fusions,CMP-Sia and GlpG were individually bound to Ni²⁺-NTA resin (1 ml per 1mg of GFP fusion) pre-equilibrated with Buffer A (PBS, 10 mM ImidazolepH 8.0, 150 mM NaCl, 10% glycerol, 0.03% DDM) using stirred mixing for2-3 hrs. The resin was washed with 10 CV of Buffer A, then 35 CV ofBuffer A supplemented with 30 mM imidazole, followed by elution using2-3 CV of Buffer A supplemented with 250 mM Imidazole.

Equal amounts of His-tagged TEV protease was added to the GFP-Hisfusions in the eluate, and the samples dialysed overnight against BufferB (20 mM Tris (pH 7.5), 150 mM NaCl, 0.03% DDM). Cleaved CMP-Sia andGlpG were isolated in the flowthrough fractions using reverse Ni²⁺-NTAbinding. Samples were concentrated to a 0.5 ml volume using centrifugalconcentrators, and submitted to a final polishing gel filtration stepusing a Superdex 200 10/300 column pre-equilibrated with Buffer B.CMP-Sia and GlpG were concentrated to 6 mg/ml and 5 mg/ml respectively,using molecular weight cut-off filters.

SQR was expressed in E. coli as an untagged construct. Membranes (˜400mg) containing SQR were resuspended in 20 mM potassium phosphate (pH7.4), 0.2 M EDTA and solubilized in 2% C₁₂E₉ for 15 min. Supernatantcontaining detergent-solubilized protein was harvested followingultracentrifugation at 100,000 g for 45 min, and filtered through a 0.2μm filter. SQR was bound to pre-equilibrated Q-sepharose Fast Flow resinin an XK26/20 column (˜24 ml). The column was washed with 2 CV of BufferC (20 mM potassium phosphate (pH 7.4), 0.2 M EDTA, 0.05% C₁₂E₉), 2 CV ofBuffer C supplemented with 100 mM NaCl, followed by elution using a(100-350) mM NaCl gradient. Fractions containing SQR were concentratedusing an Amicon stirred cell concentrator, and filtered. The SQR wasthen applied onto a Phoros 50 HQ resin using an XK16/20 column (−20 ml)pre-equilibrated with Buffer C, followed by a Sephacryl 300 26/60pre-equilibrated with buffer D (20 mM potassium phosphate (pH 7.4),0.05% C₁₂E₉). The final buffer exchange was performed on a Superdex 20010/300 gel filtration column pre-equilibrated with 20 mM Tris (pH 7.6),0.2% decyl-β-D-maltoside (DM). SQR was concentrated to 12 mg/ml usingmolecular weight cut-off filters.

Samples for CPM Assay and Gel Filtration Analysis.

CPM dye (Invitrogen), stored in DMSO (Sigma), was diluted (1:100) inBuffer B supplemented with 5 mM EDTA. Test amphiphiles or DDM were usedat CMC+0.04 wt % concentrations in 20 mM Tris (pH 7.5), 150 mM NaCl. 1μl of the purified protein; CMP-Sia (6 mg/ml), GlpG (5 mg/ml) and SQR(12 mg/ml) was individually added to test buffers (150 μl) in Greiner96-well plates, and left for equilibration at RT for 5 min, beforeadding 3 μl diluted CPM dye. For gel filtration analysis, 10 of purifiedCMP-Sia (6 mg/ml) was diluted in 1000 μl test buffer. Test buffer (20 mMTris (pH 7.5), 150 mM NaCl) included DDM or GDN at 0.042 wt %(corresponds to CMC+0.033 wt % for DDM and CMC+0.040 wt % for GDN). 500μl aliquots of the diluted protein were applied onto a Superdex 20030/100 gel filtration column, before and after incubation at 30° C. for2 hr. The column was pre-equilibrated with the respective test bufferprior to sample loading.

LeuT Functional Assay.

LeuT activity was measured according to the reported procedure (Chae etal., J. Am. Chem. Soc. 2010, 132, 16750-16752). The wild type of theleucine transporter (LeuT) from Aquifex aeolicus was expressed in E.coli C41(DE3) harboring pET16b encoding LeuT WT-His8, essentially asdescribed by Chae et al. Plasmid was kindly provided by E. Gouaux(Vollum Institute, Portland, Oreg., USA). Briefly, after isolation ofbacterial membranes followed by solubilization in 1% DDM, the LeuT waspurified by nickel affinity chromatography in 20 mM Tris-HCl (pH 8.0), 1mM NaCl, 199 mM KCl, 0.05% DDM. Subsequently, approx. 1.5 mg/ml proteinstock was diluted ten-fold in same buffer without DDM, but containingGDN, GLC-1, GLC-2 or GLC-3 in final concentrations of CMC+0.04 wt % orCMC+0.2 wt %, respectively. In control samples, DDM was used at theabove-mentioned final concentrations. Following protein storage at RT,at the indicated time points, samples were centrifuged and the proteinconcentration was assessed by determining absorbance at 280 nm.Concomitantly, for the corresponding time points, [³H]-Leu binding wasmeasured using scintillation proximity assay (SPA). Briefly, SPAreaction mixture consisted of 5 μL from the respective protein samples,20 nM [³H]-Leu and copper chelate (His-Tag) YSi beads (both fromPerkinElmer, USA). Binding was assessed in 200 mM NaCl in the presenceof tested compounds at the above-mentioned concentrations, and monitoredusing MicroBeta liquid scintillation counter (PerkinElmer).

β2AR-T4L Thermostability.

A receptor fusion protein of T4 lysozyme inserted in the 3^(rd)intracellular loop of the β₂AR⁴ was cloned into BestBac baculovirus(Expression Systems, CA) and expressed in Sf9 insect cell cultures. Thereceptor was solubilized and purified in DDM as previously described(Kobilka, Anal. Biochem. 1995, 231, 269-271). Briefly, the receptor waspurified in a three step procedure, M1 FLAG antibody affinitychromatography followed by alprenolol-Sepharose chromatography ending ina second M1 chromatography step. The fluorescent inverse agonistcarazolol was bound to the receptor on the second M1 resin followingextensive washing in buffer (0.1% DDM, 100 mM NaCl, 20 mM HEPES, pH 7.5)containing 30 μM carazolol. The eluted and carazolol-bound receptor wasdialyzed against buffer containing 1 μM carazolol to reduce freecarazolol concentration. The receptor was spin concentrated to 7 mg/ml(≈140 μM).

For stability measurements the carazolol-bound receptor was dilutedbelow the CMC for DDM by adding 3 μL of the concentrated receptor in aquartz cuvette containing 600 μL buffer (100 mM NaCl, 20 mM HEPES, pH7.5) with amphiphiles at various concentrations above their CMC. Thecuvette was placed in a Spex FluoroMax-3 spectrofluorometer (Jobin YvonInc.) under Peltier temperature control. Fluorescence emission fromcarazolol was obtained following 5 min incubations from 25 to 85° C. intwelve continuous 5° C. increments. Excitation was set at 325 nm, andemission was measured from 335 to 400 nm with an integration time of 0.3s nm-1 using a bandpass of 1 nm for both excitation and emission. The341:356 nm peak ratio was calculated and graphed using Microsoft Exceland GraphPad Prism software.

Solubilization and Stability Assay of β₂AR WT.

A gene encoding amino-terminally FLAG epitope tagged β₂AR was expressedin Sf9 cells by baculovirus, with no ligand present during culture.Cells were infected at a density of 4×10⁶ cells/mL and then cultured for48 hours prior to harvesting by centrifugation. Cells were resuspendedand lysed by osmotic shock with a low ionic strength buffer (20 mM TrispH 7.5, 1 mM EDTA). The lysed cells were aliquoted 35 mg per aliquot,then frozen. For extraction tests, 300 μL of solubilization buffer (20mM HEPES pH 7.5, 100 mM NaCl) containing each amphiphile was added toeach aliquot, which was then homogenized by pipet followed by grindingwith a glass dounce tissue homogenizer. After two-hour incubation at 4°C., samples were centrifuged at maximum speed in a tabletopmicrocentrifuge to pellet insoluble material. Supernatant was removedand assayed for protein concentration by DC protein assay (Bio-Rad).

The amount of functional receptor was quantified by incubation for 1hour with 10 nM 3H dihydroalprenolol. Samples were then separated by gelfiltration over G-50 resin and radioactivity was quantified by liquidscintillation. Nonspecific binding was measured in the presence of 10 μMalprenolol. Assays were performed in triplicate at time pointsindicated. G-50 filtration was performed in buffer containing 20 mMHEPES pH 7.5, 100 mM NaCl, 10-fold CMC of the detergent tested. Allbinding assays were performed with ice cold buffers.

Solubilization of 6-Opioid Receptor-T4L Fusion (δOR-T4L).

FLAG epitope tagged 60R-T4L was expressed in Sf9 insect cells usingbaculovirus particles generated by the pFastBac vector system(Invitrogen). Insect cells were infected and cultured as for the β₂ARand cells were lysed by osmotic shock as done for cells expressing β₂AR.Lysed cells were used for extraction tests by adding 40 mg of cells to200 μL of solubilization buffer (20 mM HEPES pH 7.5, 100 mM NaCl)containing each amphiphile. Cell membranes were homogenized insolubilization buffer by 20 passes through a narrow bore needle coupledto a 1 ml syringe. Solubilization reactions were then incubated at 4° C.for two hours and then centrifuged at maximum speed in a tabletopmicrocentrifuge. The amount of functional receptor after solubilizationwas quantified by incubation for 1 hour with 10 nM 3H diprenorphine.Binding assays using gel-filtration were carried out as for β₂AR, withthe exception that 10 μM naloxone was used to determine nonspecificbinding.

Solubilization and Thermostability Assay of MelB.

The reported protocol3 was used to evaluate MelB stability with DDM andGDN. Vector pK35ΔAHB/WT MelB/CH6 encoding the wild-type MelB with a6-His tag at the C-terminus and E. coli DW2-R cells (ΔmelB and ΔlacZY)are used for the assay. Cells were harvested, resuspended in a buffercontaining 20 mM Tris, pH 7.5, 200 mM NaCl and 10% glycerol. Theharvested cells were subjected to French press and centrifugation at20,000 g for 15 min. Subsequently, membranes were obtained viaultracentrifugation at 43,000 rpm for 3 hr in the Beckman rotor, Type 45Ti rotor. A protein assay was carried out with a BCA kit (ThermoScientific, Rockford, Ill.). For the measurement of solubilizationefficiency, membrane samples containing MelB were incubated with asolubilization buffer (20 mM Tris, 200 mM NaCl, 10% glycerol, 20 mMmelibiose, pH, 7.5) and 1.5 wt % DDM or GDN at 0° C. for 10 min. Thefinal protein concentration was 10 mg/mL. For the MelB thermostability,the samples were incubated for 90 min at the four different temperatures(0, 45, 55, and 65° C.). After ultracentrifugation at 355,590 g in aBeckman Optima™ MAX Ultracentrifuge using a TLA-100 rotor for 45 min at4° C., 10 μg protein before and after spin for each condition wasanalyzed by SDS-12% PAGE and immunoblotted with Penta-His-HRP antibody(Qiagen, Germantown, Md.).

CMP-Sia Solubilization.

CMP-Sia was expressed as a fusion protein with a C-terminal GFP inFGY217 Saccharomyces cerevisiae cells. Cell lysis was conducted by usinga cell disruptor (Constant Systems) and the protein samples weresubjected to centrifugation at 15,000 g for 10 mins to remove unbrokencells and debris. Subsequently, the membranes were harvested byultracentrifugation at 150,000 g for 45 min. The membranes wereresuspended in 50 mM Tris-HCl (pH 7.6), 1 mM EDTA, 0.6 M sorbitol andthe protein concentration was estimated using a BCA kit (Pierce). Themembranes were incubated with OG or DDM at 1.0%, or GDN at 2.0% for 1 hrat 4° C. A fluorescence value was measured for each sample before andafter ultracentrifugation at 150,000 g for 1 h. The solubilizationefficiency was calculated via the fluorescence measurements of thesoluble supernatant/the total sample.

While specific embodiments have been described above with reference tothe disclosed embodiments and examples, such embodiments are onlyillustrative and do not limit the scope of the invention. Changes andmodifications can be made in accordance with ordinary skill in the artwithout departing from the invention in its broader aspects as definedin the following claims.

All publications, patents, and patent documents are incorporated byreference herein, as though individually incorporated by reference. Theinvention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

What is claimed is:
 1. A compound of Formula V:

wherein L is —(CH₂)_(n)— where n is 1-12, or a direct bond; X is adirect bond, NH or O; Y is O or absent; Z is H, methyl, ethyl, propyl,or butyl; the dashed line represents an optionally present double bond;and each Sac is independently an oxygen-linked monosaccharide,disaccharide, or trisaccharide.
 2. The compound of claim 1 wherein theoptional double bond is present.
 3. The compound of claim 1 wherein eachSac is an oxygen-linked monosaccharide.
 4. The compound of claim 1wherein each Sac is an oxygen-linked disaccharide.
 5. The compound ofclaim 1 wherein each Sac is an oxygen-linked trisaccharide.
 6. Thecompound of claim 1 wherein X is NH, Y is O, Z is H, and L is a directbond.
 7. The compound of claim 1 wherein L is —CH₂—, X is O, Y isabsent, and Z is Me.
 8. The compound of claim 1 wherein L is a directbond, X is a direct bond, Y is absent, and Z is H.
 9. The compound ofclaim 1 wherein the compound is a compound of Formula VI:

wherein DiSac is an oxygen-linked disaccharide.
 10. The compound ofclaim 9 wherein the compound is:


11. A composition comprising a compound of claim 1 and an isolatedmembrane protein.
 12. A micelle comprising a compound of claim
 1. 13.The micelle of claim 12, further comprising a polypeptide or a protein.14. A method of solubilizing or stabilizing a membrane proteincomprising contacting a membrane protein with an effective amount of acompound as described by claim 1, in an aqueous solution, and optionallyheating the protein and the compound, thereby forming a solubilized orstabilized aggregation or micelle.
 15. The method of claim 14,comprising heating the protein and the compound, thereby forming amicelle.
 16. A method of extracting a protein from a lipid bilayercomprising contacting the lipid bilayer with an effective amount of acompound of claim 1 in an aqueous solution to form a mixture, optionallyin the presence of a buffer, thereby forming an aggregation of thecompound and the membrane protein extracted from the lipid bilayer, andseparating the aggregation from the mixture.